Nucleic acid ligands which bind to hepatocyte growth factor/scatter factor (HGF/SF) or its receptor c-met

ABSTRACT

The invention provides nucleic acid ligands to hepatocyte growth factor/scatter factor (HGF) and its receptor c-met. The nucleic acid ligands of the instant invention are isolated using the SELEX method. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The nucleic acid ligands of the invention are useful as diagnostic and therapeutic agents for diseases in which elevated HGF and c-met activity are causative factors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/364,539, filed Jul. 29, 1999, entitled “Nucleic Acid Ligandswhich Bind to Hepatocyte Growth Factor Scatter Factor (HGF/SF) or itsReceptor C-Met,” which is a continuation-in-part of U.S. patentapplication Ser. No. 09/502,344, filed Aug. 27, 1998, entitled “NucleicAcid Ligands,” which is a continuation of U.S. patent application Ser.No. 08/469,609, filed Jun., 6, 1995, entitled “Method for Detecting aTarget Molecule in a Sample Using a Nucleic Acid Ligand,” now U.S. Pat.No. 5,843,653, which is a continuation of U.S. patent application Ser.No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,”now U.S. Pat. No. 5,475,096, which is a continuation-in-part of U.S.patent application Ser. No. 07/536,428, filed Jun. 11, 1990, nowabandoned.

FIELD OF THE INVENTION

[0002] This invention is directed towards obtaining nucleic acid ligandsof hepatocyte growth factor/scatter factor (HGF) and its receptor c-met.The method used in the invention is called SELEX, which is an acronymfor Systematic Evolution of Ligands by EXponential enrichment. Theinvention is also directed towards therapeutic and diagnostic reagentsfor diseases in which elevated HGF and c-met activity are causativefactors.

BACKGROUND OF THE INVENTION

[0003] Hepatocyte growth factor/scatter factor (abbreviated herein asHGF) is a potent cytokine which, through interaction with its receptorc-met, stimulates proliferation, morphogenesis, and migration of a widevariety of cell types, predominantly epithelial. HGF and c-met areinvolved in several cellular processes involved in tumorigenesis,notably angiogenesis and motogenesis, the latter having been implicatedin the migration of cells required for metastasis (reviewed inreferences Jiang and Hiscox 1997, Histol Histopathol. 12:537-55;Tamagnone and Comoglio 1997, Cytokine Growth Factor Rev. 8:129-42;Jiang, Hiscox et al. 1999, Crit Rev Oncol Hematol. 29:209-48).Interestingly, proteases that degrade the extracellular matrix alsoactivate HGF, which in turn up-regulates urokinase type plasminogenactivator (uPA) and its receptor, resulting in an activating loopfeeding the invasive and migratory processes required for metastaticcancer.

[0004] HGF and the c-met receptor are expressed at abnormally highlevels in a large variety of solid tumors. In addition to numerousdemonstrations in vitro of the effects of HGF/c-met on the behavior oftumor cell lines, the levels of HGF and/or c-met have been measured inhuman tumor tissues (reviewed in reference Jiang 1999, Crit Rev OncolHematol. 29:209-48). High levels of HGF and/or c-met have been observedin liver, breast, pancreas, lung, kidney, bladder, ovary, brain,prostate, gallbladder and myeloma tumors in addition to many others.

[0005] For several of the cancer types listed above, the prognosticvalue of measuring HGF/c-met levels has been evaluated and found to bepotentially useful for determining the progression and severity ofdisease. The correlative data are strongest in the case of breast cancer(Ghoussoub, Dillon et al. 1998, Cancer. 82:1513-20; Toi, Taniguchi etal. 1998, Clin Cancer Res. 4:659-64), and non-small cell lung cancer(Siegfried, Weissfeld et al. 1997, Cancer Res. 57:433-9; Siegfried,Weissfeld et al. 1998, Ann Thorac Surg. 66:1915-8).

[0006] Elevated levels of HGF and c-met have also been observed innon-oncological settings, such as hypertension (Morishita, Aoki et al.1997, J Atheroscler Thromb. 4:12-9; Nakamura, Moriguchi et al. 1998,Biochem Biophys Res Commun. 242:238-43), arteriosclerosis (Nishimura,Ushiyama et al. 1997, J Hypertens. 15:1137-42; Morishita, Nakamura etal. 1998, J Atheroscler Thromb. 4:128-34), myocardial infarction (Sato,Yoshinouchi et al. 1998, J Cardiol. 32:77-82), and rheumatoid arthritis(Koch, Halloran et al. 1996, Arthritis Rheum. 39:1566-75), raising thepossibility of additional therapeutic and diagnostic applications.

[0007] The role of HGF/c-met in metastasis has been elucidated in miceusing cell lines transformed with HGF/c-met (reviewed in referenceJeffers, Rong et al. 1996, J Mol Med. 74:505-13). In another metastasismodel, human breast carcinoma cells expressing HGF/c-met were injectedin the mouse mammary fat pad, resulting in eventual lung metastases inaddition to the primary tumor (Meiners, Brinkmann et al. 1998, Oncogene.16:9-20). Also, transgenic mice which overexpress HGF become tumor-ladenat many loci (Takayama, LaRochelle et al. 1997, Proc Natl Acad Sci U SA. 94:701-6).

[0008] None of the data mentioned above provide proof of a directcausative role of HGF/c-met in human cancer, although the accumulatedweight of the correlative data are convincing. However, a causalconnection was established between germ-line c-met mutations, whichconstitutively activate its tyrosine kinase domain, and the occurrenceof human papillary renal carcinoma (Schmidt, Duh et al. 1997, Nat Genet.16:68-73).

[0009] Recent work on the relationship between inhibition ofangiogenesis and the suppression or reversion of tumor progression showsgreat promise in the treatment of cancer (Boehm, Folkman et al. 1997,Nature. 390:404-7). In this report, it was shown that the use ofmultiple angiogenesis inhibitors confers superior tumorsuppression/regression compared to the effect of a single inhibitor.Angiogenesis is markedly stimulated by HGF, as well as vascularendothelial growth factor (VEGF) and basic fibroblast growth factor(bFGF) (Rosen, Lamszus et al. 1997, Ciba Found Symp. 212:215-26). HGFand VEGF were recently reported to have an additive or synergisticeffect on mitogenesis of human umbilical vein endothelial cells (HUVECs)(Van Belle, Witzenbichler et al. 1998, Circulation. 97:381-90). Similarcombined effects are likely to contribute to angiogenesis andmetastasis.

[0010] Human HGF protein is expressed as a single peptide chain of 728amino acids (reviewed in references Mizuno and Nakamura 1993, Exs.65:1-29; Rubin, Bottaro et al. 1993, Biochim Biophys Acta. 1155:357-71;Jiang 1999, Crit Rev Oncol Hematol. 29:209-48). The amino-terminal 31residue signal sequence of HGF is cleaved upon export, followed byproteolytic cleavage by uPA and/or other proteases. The mature proteinis a heterodimer consisting of a 463 residue α-subunit and a 234 residueβ-subunit, linked via a single disulfide bond. HGF is homologous toplasminogen: its α-subunit contains an N-terminalplasminogen-activator-peptide (PAP) followed by four kringle domains,and the β-subunit is a serine protease-like domain, inactive because itlacks critical catalytic amino acids. The recently solved crystalstructure of an HGF fragment containing PAP and the first kringle domainindicate that this region is responsible for heparin binding anddimerization (Chirgadze, Hepple et al. 1999, Nat Struct Biol. 6:72-9),in addition to receptor interaction.

[0011] Human c-met protein is exported to the cell surface via a 23amino acid signal sequence (reviewed in references Comoglio 1993, Exs.65:131-65; Rubin 1993, Biochim Biophys Acta. 1155:357-71; Jiang 1999,Crit Rev Oncol Hematol. 29:209-48). The exported form of c-met isinitially a pro-peptide which is proteolytically cleaved. The matureprotein is a heterodimer consisting of an extracellular 50 kDa α-subunitbound by disulfide bonds to a 140 kDa β-subunit. In addition to itsextracellular domain, the β-subunit has a presumed membrane-spanningsequence and a 435 amino acid intracellular domain containing a typicaltyrosine kinase.

[0012] HGF is produced primarily by mesenchymal cells, while c-met ismainly expressed on cells of epithelial origin. HGF is very highlyconserved at the amino acid level between species. This homology extendsinto the functional realm as observed in mitogenic stimulation ofhepatocytes in culture by HGF across species, including human, rat,mouse, pig and dog. This indicates that human HGF can be usedcross-specifically in a variety of assays.

[0013] Given the roles of HGF and c-met in disease, it would bedesirable to have agents that bind to and inhibit the activity of theseproteins. It would also be desirable to have agents that can quantitatethe levels of HGF and c-met in individual in order to gather diagnosticand prognostic information.

[0014] The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by EXponential enrichment, termed the SELEX process, it hasbecome clear that nucleic acids have three dimensional structuraldiversity not unlike proteins. The SELEX process is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules and is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment,” now abandoned, U.S. Pat. No.5,475,096 entitled “Nucleic Acid Ligands”, U.S. Pat. No. 5,270,163 (seealso WO 91/19813) entitled “Methods for Identifying Nucleic AcidLigands,”each of which is specifically incorporated by reference herein.Each of these applications, collectively referred to herein as the SELEXPatent Applications, describes a fundamentally novel method for making anucleic acid ligand to any desired target molecule. The SELEX processprovides a class of products which are referred to as nucleic acidligands or aptamers, each having a unique sequence, and which has theproperty of binding specifically to a desired target compound ormolecule. Each SELEX-identified nucleic acid ligand is a specific ligandof a given target compound or molecule. The SELEX process is based onthe unique insight that nucleic acids have sufficient capacity forforming a variety of two- and three-dimensional structures andsufficient chemical versatility available within their monomers to actas ligands (form specific binding pairs) with virtually any chemicalcompound, whether monomeric or polymeric. Molecules of any size orcomposition can serve as targets. The SELEX method applied to theapplication of high affinity binding involves selection from a mixtureof candidate oligonucleotides and step-wise iterations of binding,partitioning and amplification, using the same general selection scheme,to achieve virtually any desired criterion of binding affinity andselectivity. Starting from a mixture of nucleic acids, preferablycomprising a segment of randomized sequence, the SELEX method includessteps of contacting the mixture with the target under conditionsfavorable for binding, partitioning unbound nucleic acids from thosenucleic acids which have bound specifically to target molecules,dissociating the nucleic acid-target complexes, amplifying the nucleicacids dissociated from the nucleic acid-target complexes to yield aligand-enriched mixture of nucleic acids, then reiterating the steps ofbinding, partitioning, dissociating and amplifying through as manycycles as desired to yield highly specific high affinity nucleic acidligands to the target molecule.

[0015] It has been recognized by the present inventors that the SELEXmethod demonstrates that nucleic acids as chemical compounds can form awide array of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby nucleic acids in biological systems.

[0016] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796, both entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” describe the use of the SELEX process inconjunction with gel electrophoresis to select nucleic acid moleculeswith specific structural characteristics, such as bent DNA. U.S. patentapplication Ser. No. 08/123,935, filed Sep. 17, 1993, entitled“Photoselection of Nucleic Acid Ligands,” now abandoned, U.S. Pat. No.5,763,177 entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX”and U.S. patent application Ser. No. 09/093,293, filed Jun. 8, 1998,entitled “Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” describe aSELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, which can be non-peptidic, termed Counter-SELEX. U.S.Pat. No. 5,567,588 entitled “Systematic Evolution of Ligands byEXponential Enrichment: Solution SELEX,” describes a SELEX-based methodwhich achieves highly efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule.

[0017] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985 entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

[0018] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459 entitled “SystematicEvolution of Ligands by EXponential Enrichment: Chimeric SELEX,” andU.S. Pat. No. 5,683,867 entitled “Systematic Evolution of Ligands byEXponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0019] The SELEX method further encompasses combining selected nucleicacid ligands with lipophilic compounds or non-immunogenic, highmolecular weight compounds in a diagnostic or therapeutic complex asdescribed in U.S. patent application Ser. No. 08/434,465, filed May 4,1995, entitled “Nucleic Acid Ligand Complexes”. Each of the abovedescribed patent applications which describe modifications of the basicSELEX procedure are specifically incorporated by reference herein intheir entirety.

[0020] It is an object of the present invention to obtain nucleic acidligands to HGF and c-met using the SELEX process.

[0021] It is a further object of the invention to obtain nucleic acidligands that act as inhibitors of HGF and c-met.

[0022] It is a further object of the invention to provide therapeuticand diagnostic agents for tumorigenic conditions in which HGF and c-metare implicated.

[0023] It is yet a further object of the invention to use nucleic acidligands to HGF and c-met to diagnose and treat hypertension,arteriosclerosis, myocardial infarction, and rheumatoid arthritis.

[0024] It is an even further object of the invention to use nucleic acidligands to HGF singly or in combination with other nucleic acid ligandsthat inhibit VEGF and/or bFGF, and/or possibly other angiogenesisfactors.

SUMMARY OF THE INVENTION

[0025] Methods are provided for generating nucleic acid ligands to HGFand c-met. The methods use the SELEX process for ligand generation. Thenucleic acid ligands provided by the invention are useful as therapeuticand diagnostic agents for a number of diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 illustrates the template and primer oligonucleotides used2′-F-pyrimidine RNA SELEX experiments. The 5′ fixed region of thetemplate and primers contains a T7 promoter to facilitate transcriptionof RNA by T7 RNA polymerase.

[0027]FIG. 2 illustrates RNaseH cleavage primers used in hybridizationtruncate SELEX. Bases depicted in bold-type are 2′-O-methyl modified andbases underlined are deoxyribonucleosides. The random region isdesignated as “N”. Upon treatment with RNaseH, the fixed regions areremoved at the positions indicated by the carets. Note that the thereare two possible cleavage sites at the 5-prime end of the fixed region,resulting in RNA which has one or two fixed G residues.

[0028]FIG. 3 illustrates binding of SELEX pools to HGF. FIG. 3A showsHGF SELEX 1 30N7 pools. FIG. 3B shows HGF SELEX 2 30N8 pools.

[0029]FIG. 4 illustrates two methods of evaluating HGF SELEX 3 30N7 poolbinding to HGF. In

[0030]FIG. 4A, heparin competes with RNA pools for binding to 2.7 nMHGF. FIG. 4B illustrates conventional pool binding.

[0031]FIG. 5 illustrates two methods of evaluating HGF SELEX 3 30N7 poolbinding to HGF.

[0032]FIG. 5A shows that tRNA competes with RNA pools for binding to 2.7nM HGF.

[0033]FIG. 5B shows conventional pool binding.

[0034]FIG. 6 illustrates inhibition of 10 ng/ml HGF stimulation ofstarved HUVECs by aptamers.

[0035]FIG. 6A shows a 1st set of aptamers. FIG. 6B illustrates a 2nd setof aptamers.

[0036]FIG. 7 illustrates truncates of aptamer 8-102. FIG. 7A showspredicted two-dimensional structures of full-length and truncatedsequences. FIG. 7B shows binding of full-length and truncated aptamersto HGF.

[0037]FIG. 8 illustrates truncates of aptamer 8-17. FIG. 8A shows apredicted two-dimensional structures of full-length and truncatedsequences. FIG. 8B shows binding of full-length and truncated aptamersto HGF.

[0038]FIG. 9 illustrates binding of HGF truncate SELEX pools. FIG. 9Ashows the HGF SELEX 4 30N7 series. FIG. 9B shows the HGF SELEX 5 30N7series.

[0039]FIG. 10 shows aptamer inhibition of 100 ng/ml HGF stimulation of4MBr-5 cells.

[0040]FIG. 11 illustrates aptamer inhibition of 50 ng/ml HGF stimulationof 4MBr5 cells.

[0041]FIG. 11A shows the effect of PEGylation of 36 mer. FIG. 11B showsa comparison of PEGylated 36 mer to best full-length inhibitor 8-17.

[0042]FIG. 12 shows aptamer inhibition of 50 ng/ml HGF stimulation of4MBr-5 cells.

[0043]FIG. 13 shows HUVEC mitogenesis by 10 ng/ml HGF, 10 ng/ml VEGF, orboth HGF and VEGF.

[0044]FIG. 14 illustrates aptamer-mediated inhibition of HUVECmitogenesis. FIG. 14A shows stimulation by both HGF and VEGF inhibitedby either HGF or VEGF aptamers or both.

[0045]FIG. 14B illustrates stimulation by HGF alone inhibited by eitherHGF or VEGF aptamer or both. FIG. 14C illustrates stimulation by VEGFalone inhibited by either HGF or VEGF aptamer or both.

[0046]FIG. 15 depicts ratios of selected to unselected partially2′-O-methyl substituted purines in aptamer NX22354.

[0047]FIG. 16 illustrates 2′-O-methyl substituted derivatives of NX22354binding to HGF: average of two experiments.

[0048]FIG. 17 illustrates binding of SELEX pools to c-met. FIG. 17Ashows c-Met SELEX 40N7. FIG. 17B shows c-Met SELEX 30N8. FIG. 17C showsboth SELEXes: a, c pools, 40N7; b, d pools, 30N8.

[0049]FIG. 18 illustrates binding of c-met SELEX pools to c-met and KDRIg fusion proteins.

[0050]FIG. 19 shows binding of c-met 40N7 cloned aptamers to c-met andKDR Ig fusion proteins. FIG. 19A shows clone 7c-1. FIG. 19B showsclone7c-3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The central method utilized herein for identifying nucleic acidligands to HGF and c-met is called the SELEX process, an acronym forSystematic Evolution of Ligands by Exponential enrichment and involves(a) contacting the candidate mixture of nucleic acids with HGF or c-met,or expressed domains or peptides corresponding to HGF or c-met, (b)partitioning between members of said candidate mixture on the basis ofaffinity to HGF or c-met, and c) amplifying the selected molecules toyield a mixture of nucleic acids enriched for nucleic acid sequenceswith a relatively higher affinity for binding to HGF or c-met.

[0052] Definitions

[0053] Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided:

[0054] As used herein, “nucleic acid ligand” is a non-naturallyoccurring nucleic acid having a desirable action on a target. Nucleicacid ligands are often referred to as “aptamers”. The term aptamer isused interchangeably with nucleic acid ligand throughout thisapplication. A desirable action includes, but is not limited to, bindingof the target, catalytically changing the target, reacting with thetarget in a way which modifies/alters the target or the functionalactivity of the target, covalently attaching to the target as in asuicide inhibitor, facilitating the reaction between the target andanother molecule. In the preferred embodiment, the action is specificbinding affinity for a target molecule, such target molecule being athree dimensional chemical structure other than a polynucleotide thatbinds to the nucleic acid ligand through a mechanism which predominantlydepends on Watson/Crick base pairing or triple helix binding, whereinthe nucleic acid ligand is not a nucleic acid having the knownphysiological function of being bound by the target molecule. In thepresent invention, the targets are c-met and HGF or portions thereof.Nucleic acid ligands include nucleic acids that are identified from acandidate mixture of nucleic acids, said nucleic acid ligand being aligand of a given target, by the method comprising: a) contacting thecandidate mixture with the target, wherein nucleic acids having anincreased affinity to the target relative to the candidate mixture maybe partitioned from the remainder of the candidate mixture; b)partitioning the increased affinity nucleic acids from the remainder ofthe candidate mixture; and c) amplifying the increased affinity nucleicacids to yield a ligand-enriched mixture of nucleic acids.

[0055] As used herein, “candidate mixture” is a mixture of nucleic acidsof differing sequence from which to select a desired ligand. The sourceof a candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

[0056] As used herein, “nucleic acid” means either DNA, RNA,single-stranded or double-stranded, and any chemical modificationsthereof. Modifications include, but are not limited to, those whichprovide other chemical groups that incorporate additional charge,polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

[0057] “SELEX” methodology involves the combination of selection ofnucleic acid ligands which interact with a target in a desirable manner,for example binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain nucleic acidligands to HGF and c-met.

[0058] The SELEX methodology is described in the SELEX PatentApplications.

[0059] “SELEX target” or “target” means any compound or molecule ofinterest for which a ligand is desired. A target can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc.without limitation. In this application, the SELEX targets are HGF andc-met. In particular, the SELEX targets in this application includepurified HGF and c-met, and fragments thereof, and short peptides orexpressed protein domains comprising HGF or c-met. Also includes astargets are fusion proteins comprising portions of HGF or c-met andother proteins.

[0060] As used herein, “solid support” is defined as any surface towhich molecules may be attached through either covalent or non-covalentbonds. This includes, but is not limited to, membranes, microtiterplates, magnetic beads, charged paper, nylon, Langmuir-Bodgett films,functionalized glass, germanium, silicon, PTFE, polystyrene, galliumarsenide, gold, and silver. Any other material known in the art that iscapable of having functional groups such as amino, carboxyl, thiol orhydroxyl incorporated on its surface, is also contemplated. Thisincludes surfaces with any topology, including, but not limited to,spherical surfaces and grooved surfaces.

[0061] As used herein, “HGF” refers to hepatocyte growth factor/scatterfactor. This includes purified hepatocyte growth factor/scatter factor,fragments of hepatocyte growth factor/scatter factor, chemicallysynthesized fragments of hepatocyte growth factor/scatter factor,derivatives or mutated versions of hepatocyte growth factor/scatterfactor, and fusion proteins comprising hepatocyte growth factor/scatterfactor and another protein. “HGF” as used herein also includeshepatocyte growth factor/scatter factor isolated from species other thanhumans.

[0062] As used herein “c-met” refers to the receptor for HGF. Thisincludes purified receptor, fragments of receptor, chemicallysynthesized fragments of receptor, derivatives or mutated versions ofreceptor, and fusion proteins comprising the receptor and anotherprotein. “c-met” as used herein also includes the HGF receptor isolatedfrom a species other than humans.

[0063] Note that throughout this application, various references arecited. Every reference cited herein is specifically incorporated in itsentirety.

[0064] A. Preparing Nucleic Acid Ligands to HGF and C-met

[0065] In the preferred embodiment, the nucleic acid ligands of thepresent invention are derived from the SELEX methodology. The SELEXprocess is described in U.S. patent application Ser. No. 07/536,428,entitled Systematic Evolution of Ligands by Exponential Enrichment, nowabandoned, U.S. Pat. No. 5,475,096 entitledNucleic Acid Ligands, andU.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled Methods forIdentifying Nucleic Acid Ligands. These applications, each specificallyincorporated herein by reference, are collectively called the SELEXPatent Applications.

[0066] The SELEX process provides a class of products which are nucleicacid molecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

[0067] In its most basic form, the SELEX process may be defined by thefollowing series of steps:

[0068] 1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are chosen either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0069] 2) The candidate mixture is contacted with the selected targetunder conditions favorable for binding between the target and members ofthe candidate mixture. Under these circumstances, the interactionbetween the target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

[0070] 3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

[0071] 4) Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

[0072] 5) By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer uniquesequences, and the average degree of affinity of the nucleic acids tothe target will generally increase. Taken to its extreme, the SELEXprocess will yield a candidate mixture containing one or a small numberof unique nucleic acids representing those nucleic acids from theoriginal candidate mixture having the highest affinity to the targetmolecule.

[0073] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796 both entitled “Method for Selecting Nucleic Acids on the Basisof Structure,” describe the use of the SELEX process in conjunction withgel electrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,”, now abandoned, U.S. Pat. No. 5,763,177 entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S.patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” all describea SELEX based method for selecting nucleic acid ligands containingphotoreactive groups capable of binding and/or photocrosslinking toand/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific nucleic acid ligands able to discriminate between closelyrelated molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588entitled “Systematic Evolution of Ligands by Exponential Enrichment:Solution SELEX,” describes a SELEX-based method which achieves highlyefficient partitioning between oligonucleotides having high and lowaffinity for a target molecule. U.S. Pat. No. 5,496,938 entitled“Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describes methods forobtaining improved nucleic acid ligands after SELEX has been performed.U.S. Pat. No. 5,705,337 entitled “Systematic Evolution of Ligands byExponential Enrichment: Chemi-SELEX,” describes methods for covalentlylinking a ligand to its target.

[0074] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985 entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,637,459, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

[0075] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459 entitled “SystematicEvolution of Ligands by Exponential Enrichment: Chimeric SELEX,” andU.S. Pat. No. 5,683,867 entitled “Systematic Evolution of Ligands byExponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0076] In U.S. Pat. No. 5,496,938 methods are described for obtainingimproved nucleic acid ligands after the SELEX process has beenperformed. This patent, entitled Nucleic Acid Ligands to HIV-RT andHIV-1 Rev, is specifically incorporated herein by reference.

[0077] One potential problem encountered in the diagnostic use ofnucleic acids is that oligonucleotides in their phosphodiester form maybe quickly degraded in body fluids by intracellular and extracellularenzymes such as endonucleases and exonucleases before the desired effectis manifest. Certain chemical modifications of the nucleic acid ligandcan be made to increase the in vivo stability of the nucleic acid ligandor to enhance or to mediate the delivery of the nucleic acid ligand.See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 8,1993, now abandoned, and U.S. Pat. No. 5,660,985, both entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides”, and theU.S. Patent Application entitled “Transcription-free SELEX”, U.S. patentapplication Ser. No. 09/362,578, filed Jul. 28, 1999, each of which isspecifically incorporated herein by reference. Modifications of thenucleic acid ligands contemplated in this invention include, but are notlimited to, those which provide other chemical groups that incorporateadditional charge, polarizability, hydrophobicity, hydrogen bonding,electrostatic interaction, and fluxionality to the nucleic acid ligandbases or to the nucleic acid ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,phosphorothioate or alkyl phosphate modifications, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping. In preferred embodiments of the instantinvention, the nucleic acid ligands are RNA molecules that are 2′-fluoro(2′-F) modified on the sugar moiety of pyrimidine residues.

[0078] The modifications can be pre- or post-SELEX processmodifications. Pre-SELEX process modifications yield nucleic acidligands with both specificity for their SELEX target and improved invivo stability. Post-SELEX process modifications made to 2′-OH nucleicacid ligands can result in improved in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand.

[0079] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX process (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

[0080] The nucleic acid ligands of the invention are prepared throughthe SELEX methodology that is outlined above and thoroughly enabled inthe SELEX applications incorporated herein by reference in theirentirety. The SELEX process can be performed using purified HGF orc-met, or fragments thereof as a target. Alternatively, full-length HGFor c-met, or discrete domains of HGF or c-met, can be produced in asuitable expression system. Alternatively, the SELEX process can beperformed using as a target a synthetic peptide that includes sequencesfound in HGF or c-met. Determination of the precise number of aminoacids needed for the optimal nucleic acid ligand is routineexperimentation for skilled artisans.

[0081] In some embodiments, the nucleic acid ligands become covalentlyattached to their targets upon irradiation of the nucleic acid ligandwith light having a selected wavelength. Methods for obtaining suchnucleic acid ligands are detailed in U.S. patent application Ser. No.08/123,935, filed Sep. 17, 1993, entitled “Photoselection of NucleicAcid Ligands,”, now abandoned, U.S. Pat. No. 5,763,177 entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S.patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled“Systematic Evolution of Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” each of whichis specifically incorporated herein by reference in its entirety.

[0082] In preferred embodiments, the SELEX process is carried out usingHGF or c-met attached to a solid support. A candidate mixture of singlestranded RNA molecules is then contacted with the solid support. Inespecially preferred embodiments, the single stranded RNA molecules havea 2′-fluoro modification on C and U residues, rather than a 2′-OH group.After incubation for a predetermined time at a selected temperature, thesolid support is washed to remove unbound candidate nucleic acid ligand.The nucleic acid ligands that bind to the HGF or c-met protein are thenreleased into solution, then reverse transcribed by reversetranscriptase and amplified using the Polymerase Chain Reaction. Theamplified candidate mixture is then used to begin the next round of theSELEX process.

[0083] In the above embodiments, the solid support can be anitrocellulose filter. Nucleic acids in the candidate mixture that donot interact with the immobilized HGF or c-met can be removed from thisnitrocellulose filter by application of a vacuum. In other embodiments,the HGF or c-met target is adsorbed on a dry nitrocellulose filter, andnucleic acids in the candidate mixture that do not bind to the HGF orc-met are removed by washing in buffer. In other embodiments, the solidsupport is a microtiter plate comprised of, for example, polystyrene.

[0084] In still other embodiments, the HGF or c-met protein is used as atarget for Truncate SELEX, described in U.S. patent application Ser. No.09/275,850, filed Mar. 24, 1999, entitled “The Truncation SELEX Method”,incorporated herein by reference in its entirety.

[0085] In preferred embodiments, the nucleic acid ligands thus obtainedare assayed for their ability to inhibit the HGF/c-met interaction. Inone embodiment, this is performed by performing a cell migration assay.Certain cell types, such as A549 lung carcinoma cells, will showincreased migration through a Matrigel-coated filter insert (BectonDickinson) in the presence of HGF. Thus, the degree of inhibition of HGFactivity in the presence of an HGF or c-met nucleic acid ligand can beassayed by determining the number of cells that have migrated throughthe filter in the presence of HGF.

[0086] B. Methods and Compositions for Using Nucleic Acid Ligands toTreat and Diagnose Disease

[0087] Given that elevated levels of c-met and HGF are observed inhypertension, arteriosclerosis, myocardial infarction, and rheumatoidarthritis, nucleic acid ligands will serve as useful therapeutic anddiagnostic agents for these diseases. In some embodiments, inhibitorynucleic acid ligands of HGF and c-met are administered, along with apharmaceutically accepted excipient to an individual suffering from oneof these diseases. Modifications of these nucleic acid ligands are madein some embodiments to impart increased stability upon the nucleic acidligands in the presence of bodily fluids. Such modifications aredescribed and enabled in the SELEX applications cited above.

[0088] In other embodiments, nucleic acid ligands to HGF and c-met areused to measure the levels of these proteins in an individual in orderto obtain prognostic and diagnostic information. Elevated levels ofc-met and HGF are associated with tumors in the liver, breast, pancreas,lung, kidney, bladder, ovary, brain, prostrate, and gallbladder.Elevated levels of HGF and c-met are also associated with myeloma.

[0089] In other embodiments, nucleic acid ligands that inhibit theHGF/c-met interaction are used to inhibit tumorigenesis, by inhibiting,for example, angiogenesis and motogenesis.

[0090] In one embodiment of the instant invention, a nucleic acid ligandto HGF is used in combination with nucleic acid ligands to VEGF(vascular endothelial growth factor) and/or bFGF (basic fibroblastgrowth factor) to inhibit tumor metastasis and angiogenesis. The use ofmultiple nucleic acid ligands is likely to have an additive orsynergistic effect on tumor suppression. Nucleic acid ligands thatinhibit VEGF are described in U.S. Pat. Nos. 5,849, 479, 5,811,533, andU.S. patent application Ser. No. 09/156,824, filed Sep. 18, 1998, eachof which is entitled “High Affinity Oligonucleotide Ligands to VascularEndothelial Growth Factor”, and each of which is specificallyincorporated herein by reference in its entirety. Nucleic acid ligandsto VEGF are also described in U.S. Pat. No. 5,859,228, U.S. patentapplication Ser. No. 08/870,930, filed Jun. 6, 1997, U.S. patentapplication Ser. No. 08/897,351, filed Jul. 21, 1997, and U.S. patentapplication Ser. No. 09/254,968, filed Mar. 16, 1999, each of which isentitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid LigandComplexes,” and each of which is specifically incorporated by referencein its entirety. Nucleic acid ligands to bFGF are described in U.S. Pat.No. 5,639,868 entitled “High Affinity RNA ligands for Basic FibroblastGrowth Factor”, and U.S. patent application Ser. No. 08/442,423, filedMay 16, 1995, entitled “High Affinity RNA Ligands for Basic FibroblastGrowth Factor”, each of which is specifically incorporated herein byreference in its entirety.

EXAMPLES

[0091] The following examples are given by way of illustration only.They are not to be taken as limiting the scope of the invention in anyway.

[0092] Materials and Methods

[0093] In the sections below entitled “Results: HGF” and “Results:c-met”, the following materials and methods were used:

[0094] Proteins. The HGF protein and c-met-IgG₁-His₆ fusion protein,which were used in the SELEX process, and the KDR-IgG₁-His₆ proteinswere purchased from R&D Systems, Inc. (Minneapolis, Minn.). The humanc-met-IgG₁-His₆ fusion protein—described from the amino to the carboxylterminus—consists of 932 amino acids from the extracellular domains ofthe α and β chains of c-met, a factor Xa cleavage site, 231 amino acidsfrom human IgG₁ (Fc domain), and a (His)₆ tag. This protein is referredto in the text and figures as c-met. A similar fusion protein containingthe vascular endothelial growth factor receptor KDR will be referred toas KDR.

[0095] Anti-HGF monoclonal antibody MAB294 was purchased from R&DSystems, Inc. Human IgG₁ was produced in-house by stable expression fromChinese hamster ovary cells.

[0096] SELEX templates and primers. Standard SELEX templates carrying 30or 40 random nucleotides flanked by fixed regions of the N7 or N8 seriesand associated primers (FIG. 1) were used as described (Fitzwater andPolisky 1996, Methods Enzymol. 267:275-301). Truncate SELEX was done bythe hybridization method described in U.S. patent application Ser. No.09/275,850, filed Mar. 24, 1999, entitled “The Truncation SELEX Method”,incorporated herein by reference in its entirety, using RNaseH cleavageprimers (FIG. 2).

[0097] SELEX methods. Initial HGF SELEX experiments were done by twoclosely-related partitioning methods, both involving separating freefrom bound RNA on nitrocellulose filters. Conventional SELEX involvesmixing target protein and RNA library in HBSMC buffer (hepes-bufferedsaline, 25 mM hepes, 137 mM NaCl, 5 mM KCl plus 1 mM CaCl₂, 1 mM MgCl₂,pH 7.4), followed by filtration on nitrocellulose under vacuum.Maintaining vacuum, the filter is washed in buffer, followed by vacuumrelease and RNA extraction. In spot filter SELEX, the protein is appliedto a dry nitrocellulose 13 mm filter, allowed to adsorb for severalminutes, then pre-incubated in Buffer S (HBSMC buffer plus 0.02% each officoll, polyvinylpyrrolidone, and human serum albumin) for 10 minutes at37° C. to remove unbound protein. The wash buffer is removed, and thenthe RNA library is added in the same buffer, and incubated with theprotein-bound filter. The filters are washed by repeated incubations infresh buffer, followed by RNA extraction.

[0098] SELEX was initiated with between 1 and 5 nmoles of2′-fluoro-pyrimidine RNA sequence libraries containing either a 30 or 40nucleotide randomized region sequence (FIG. 1). The RNA libraries weretranscribed from the corresponding synthetic DNA templates that weregenerated by Klenow extension (Sambrook, Fritsch et al. 1989, 3 B. 12).The DNA templates were transcribed in 1 ml reactions, each containing0.25 nM template, 0.58 μM T7 RNA polymerase, 1 mM each of ATP and GTP, 3mM each of 2′-F-CTP and 2′-F-UTP, 40 mM Tris-HCl (pH 8.0), 12 mM MgCl₂,1 mM spermidine, 5 mM DTT, 0.002% Triton X-100 and 4% polyethyleneglycol (w/v) for at least 4 hours at 37° C. The full-lengthtranscription products were purified by denaturing polyacrylamide gelelectrophoresis. Radiolabeled RNA was obtained from transcriptionreactions as described above, but containing 0.2 nM ATP and 100 μCi ofα-³²P-ATP. Alternatively, radiolabeled RNA was obtained by labeling the5′-end of RNA with (α-³²P-ATP (NEN-DuPont), catalyzed by T4polynucleotide kinase (New England Biolabs). To prepare RNA containing5′-OH groups for kinase reactions, transcription reactions included 5 mMguanosine.

[0099] For conventional filter SELEX, radiolabeled RNA pools weresuspended in HBSMC buffer to which HGF protein was added, and incubatedat 37° C. for 30 minutes to 3 hours depending on the round. Bindingreactions were then filtered under suction through 0.45 μmnitrocellulose filters (Millipore), pre-wet with binding buffer. Thefilters were immediately washed with at least 5 ml of HBSMC buffer. Foreach binding reaction, a protein-minus control reaction was done inparallel in order to determine the amount of background binding to thefilters. The amount of RNA retained on the filters was quantified byCherenkov counting, and compared with the amount input into thereactions. Filter-retained RNA was extracted with phenol and chloroform,and isolated by ethanol precipitation in the presence of 1-2 μgglycogen.

[0100] The isolated RNA was subsequently used as a template for avianmyeloblastosis virus reverse transcriptase (AMV-RT, Life Sciences) toobtain cDNA. One hundred pmoles of the 3′-primer (FIG. 1) was added tothe RNA and annealed by heating for 3 minutes at 70° C., followed bychilling on ice. The 50 μl reaction contained 5 U AMV-RT, 0.4 mM each ofdNTPs, 50 mM Tris-HCI (pH 8.3), 60 mM NaCl, 6 mM Mg(OAc)₂, and 10 mMDTT, which was incubated for 45 minutes at 48° C. The cDNA was amplifiedby PCR with the 5′- and the 3′-primers (FIG. 1), and the resulting DNAtemplate was transcribed to obtain RNA for the next round of SELEX.

[0101] To minimize selection of undesirable nitrocellulose-bindingsequences, beginning in round three, we pre-soaked pools withnitrocellulose filters before incubating with the target protein. Thistreatment worked well to control background binding and helped ensurethat each SELEX round had a positive signal/noise ratio. The progress ofSELEX was monitored by nitrocellulose filter-binding analysis of theenriched pools (see below).

[0102] Truncate SELEX was performed by the hybridization methoddescribed in U.S. patent application Ser. No. 09/275,850, filed Mar. 24,1999, entitled “The Truncation SELEX Method”, incorporated herein byreference in its entirety. Briefly, 2′-F-RNA pools were body-labeledduring transcription and cleaved by RNaseH using specific cleavageprimers to remove the fixed sequences from the SELEX pool (FIG. 2). ThisRNA was then bound to target protein HGF and recovered followingpartitioning as in a conventional filter SELEX experiment. The recoveredRNA was then biotinlyated at its 3-prime end and hybridized overnightunder appropriate conditions with single-stranded fall-lengthcomplementary strand DNA obtained from the starting SELEX pool, fromwhich the RNA had been transcribed. The RNA/DNA complexes were thencaptured on streptavidin-coated magnetic beads and extensively washed toremove non-hybridized DNA. The bound DNA in the captured RNA/DNAcomplexes was then eluted by heat denaturation and amplified usingconventional SELEX PCR primers. To complete the cycle, the resulting DNAwas then used as a transcription template for generating RNA to becleaved by RNaseH, and used in the next round of truncate SELEX.

[0103] For plate SELEX, a polystyrene well was pre-blocked in 400 μl ofblocking agent for 60 minutes at 37° C. The blocking agent was removedand the desired amount of RNA in 100 μl binding buffer was added andincubated for 60 minutes at 37 ° C. White, polystyrene breakaway wells(catalog #950-2965) used for partitioning were from VWR (Denver, Colo.).The blocking agents, I-block and Superblock, were purchased from Tropix(Bedford, Mass.) and Pierce (Rockford, Ill.), respectively. Thepreadsorbtion was done to remove any nucleic acids which might bind tothe well or the blocking agent. The random and round one libraries werenot preadsorbed to plates to avoid loss of unique sequences. C-metprotein was diluted in HBSMCK (50 mM HEPES, pH 7.4, 140 mM NaCl, 3 mMKCl, 1 mM CaCl₂, 1 mM MgCl₂), and was adsorbed to polystyrene wells byincubating 100 μl of diluted protein per well for 60 minutes at 37 ° C.The wells were each washed with three 400 μl aliquots of HIT buffer(HBSMCK, 0.1% I-block, 0.05% Tween 20), and then blocked in 400 μl ofblocking agent for 60 minutes at 37 ° C. SELEX was initiated byincubating 100 μl of RNA in the protein-bound well for 60 minutes at 37° C. The RNA was removed and the wells were washed with 400 μl aliquotsof HIT buffer. Increasing numbers of washes were used in later rounds.The wells were then washed twice with 400 μl water. RNA bound to c-metwas eluted by adding 100 μl water and heating at 95 ° C. for 5 minutesand then cooled on ice, followed by reverse transcription.

[0104] Nitrocellulose filter-binding. In binding reactions, RNAconcentrations were kept as low as possible—between 1 and 20 μM—toensure equilibrium in conditions of protein excess. Oligonucleotideswere incubated for 15 minutes at 37° C. with varying amounts of theprotein in 43 μl of the binding buffer. Thirty-two microliters of eachbinding mixture placed on pre-wet 0.45 μm nitrocellulose filters undersuction. Each well was immediately washed with 0.5 ml binding buffer.The amount of radioactivity retained on the filters was quantitated byimaging. The radioactivity that bound to filters in the absence ofprotein was used for background correction. The percentage of inputoligonucleotide retained on each filter spot was plotted against thecorresponding log protein concentration. The nonlinear least squaremethod was used to obtain the dissociation constant (K_(d); referenceJellinek, Lynott et al. 1993, Proc. Natl. Acad. Sci. USA. 90:11227-31).

[0105] Competitor titration curves were generated essentially as astandard binding curve, except that the protein and RNA concentrationswere kept constant, and the competitor concentration was varied.Competitors were also added at a fixed concentration in bindingexperiments to increase stringency for purposes of comparing poolbinding affinities. In these experiments, the competitor concentrationwas chosen based on the results from the competitor titration curves.

[0106] Molecular cloning and DNA sequencing. To obtain individualsequences from the enriched pools, we cloned the PCR products from thefinal SELEX rounds using one of two blunt-end cloning kits, PerfectlyBlunt (Novagen, Madison, Wis.), or PCR-Script (Stratagene, La Jolla,Calif.). Clones were sequenced with the ABI Prism Big Dye TerminatorCycle Sequencing kit (Perkin-Elmer Applied Biosystems, Foster City,Calif.). Sequences were obtained from an automated ABI sequencer, andtext files were collated and analyzed by computer alignment andinspection.

[0107] Boundary determinations. Five-prime and 3-prime boundaries of RNAaptamers were determined by the method of partial alkaline hydrolysis asdescribed (Jellinek, Green et al. 1994, Biochemistry. 33:10450-6).

[0108] Cell assays. Standard cell culture procedures were employed inthe course of performing in vitro experiments to test aptamer-mediatedinhibition of HGF activity. For cell migration assays, monolayers ofA549 (lung carcinoma) cells were grown on the top-sides ofMatrigel-coated filter inserts (Becton Dickinson, Franklin Lakes, N.J.)in 24-well plates. The cells adhere to the upper surface of the filter,which is placed in growth medium containing HGF. After two days, thecells are physically removed from the top surface of the filter. Thefilter is then removed from the insert and stained with crystal violet.Since all cells on the top of the filter are gone, the only cells thatremain are those that are have migrated to the bottom of the filter. Inthe presence of HGF, significantly more cells are found on the bottom ofthe filter compared to controls without HGF.

[0109] Oligonucleotide synthesis and modification. RNA was routinelysynthesized by standard cyanoethyl chemistry as modified (Green,Jellinek et al. 1995, Chem Biol. 2:683-95). Two-prime-fluoro-pyrimidinephosphoramidite monomers were obtained from JBL Scientific (San LuisObispo, Calif.); 2′-OMe purine, 2′-OH purine, hexyl amine, and the dTpolystyrene solid support were obtained from Glen Research (Sterling,Va.).

[0110] For addition of 40K-PEG, RNA oligomers were synthesized with anamino-linker at the 5′-position. This was subsequently reacted withNHS-ester 40K-PEG manufactured by Shearwater Polymers, Inc. (Huntsville,Ala.), and purified by HPLC on a reverse-phase preparative column.

[0111] 2′-O-methyl purine substitution. Determination of which2′-OH-purines can be substituted by 2′-O-methyl-purine was done asdescribed (Green 1995, Chem Biol. 2:683-95). Briefly, a set ofoligonucleotides was synthesized with a mixture of 2′-O-methyl amiditesand 2′-OH amidites at defined purine positions. The set was designed sothat each oligonucleotide contains a subset of partially-substitutedpurines, and the complete set encompasses all purines. Each aptamer was5′-end labeled and subjected to limited alkaline hydrolysis followed bybinding to HGF protein at two different concentrations, 50 and 100 μM.Following binding, protein-bound RNA was separated by standardnitrocellulose filtration. Bound RNA was recovered and analyzed byhigh-resolution gel electrophoresis. The fragmented alkaline-hydrolyzedaptamers which were not exposed to HGF were run to establish thecleavage patterns of the unselected aptamers. Hydrolysis occurs only at2′-OH-purines. If a given position requires 2′-OH for optimal binding toHGF, it appears as a relatively darker band compared to the unselectedaptamer at that position.

[0112] Results—HGF

[0113] Five HGF SELEX experiments were done in total. The first threewere done by conventional filter SELEX, while the latter two were doneby the hybridization truncate SELEX method described in U.S. patentapplication Ser. No. 09/275,850, filed Mar. 24, 1999, entitled “TheTruncation SELEX Method”, incorporated herein by reference in itsentirety. HGF SELEX 1 was done with 30N7 2′-F-RNA for thirteen rounds ofconventional filter binding. HGF SELEX 2 was done with 3ON8 2′-F-RNA forthirteen rounds of conventional filter binding. HGF SELEX 3 was donewith 30N7 2′-F-RNA for seven rounds by spot filter binding, followed byeight rounds of filter binding. HGF SELEX 4 was done by ace,hybridization filter SELEX for three rounds, starting with pool 8 fromHGF SELEX 1. HGF SELEX 5 was done by hybridization filter SELEX forthree rounds, starting with pool 11 from HGF SELEX 3. HBSMC buffer wasused in conventional SELEX reactions, and in spot filter SELEX, blockingagents were added as described in Materials and Methods.

[0114] RNA pool binding with and without competitors heparin and tRNA.To evaluate SELEX progress, binding curves with purified HGF proteinwere routinely done with evolved pools during the course of theseexperiments. Representative binding curves are shown for HGF SELEXexperiments 1 and 2 (FIG. 3). These data were used to ascertain when aSELEX was complete in that further progress was not likely to occur byperforming additional rounds. HGF SELEX 1 reached its maximal binding byround 8, with a binding affinity of approximately 0.1 nM (FIG. 3A;earlier rounds and round 9 were examined in other experiments). HGFSELEX 2 reached its maximal binding by round 10, with a binding affinityof approximately 0.1 nM (FIG. 3B). HGF SELEX 3 reached its maximalbinding by round 11, after seven rounds of spot filter partitioningfollowed by four rounds of conventional filter SELEX (see FIG. 4B). ASELEX experiment which was deemed complete was characterized by cloningand sequencing (see below).

[0115] HGF, like other proteins which have large clusters of positivelycharged amino acids, exhibits a high degree of non-specific binding topolyanionic compounds. For example, random RNA pools bind to HGF withlow nanomolar affinity, similar to the value reported for HGF binding toheparin, a polyanionic sulfated polysaccharide known to have animportant biological role in HGF function (Zioncheck, Richardson et al.1995, J Biol Chem. 270:16871-8). Competition binding to heparin as wellas the non-specific competitor tRNA was done to provide an additionalmeans of evaluating SELEX progress. We did this because the binding ofrandom and evolved RNA pools to HGF occurs in a high-affinity rangewhich makes it difficult to monitor progress. In other words, random RNAbinds so well to HGF that the affinity enhancement of the evolved poolsmay not be adequately assessed in conventional binding experiments inthe absence of competitor.

[0116] RNA pools from HGF SELEX 3 were subjected to competition withheparin (FIG. 4A). This experiment demonstrates that random RNA isconsiderably more sensitive to competition for binding to HGF than arethe evolved pools. These data are compared to those obtained from abinding curve with the same three RNA pools (FIG. 4B). In the absence ofheparin competition, binding of random RNA to HGF is nearly as good asthat of the evolved pools, whereas the heparin competition reveals thatthe evolved pools are significantly different in composition from randomRNA. In addition, while rounds 8 and 11 are indistinguishable inconventional binding curves, round 11 exhibits improved binding based onincreased resistance to heparin competition. These data contributed tothe choice of round 11 as the maximally binding pool from which wecloned and sequenced.

[0117] A similar, but more pronounced, effect was observed with tRNA asthe competitor (FIG. 5A). These data indicate that the round 11 poolfrom HGF SELEX 3 are at least four orders of magnitude more resistant tocompetition for binding to HGF than is random RNA. From these curves, itwas determined that 800 nM tRNA is the maximum concentration at whichcomplete binding of evolved RNA persists. Therefore, binding curves weredone at this tRNA concentration to compare the binding of differentevolved pools (FIG. 5B). These curves were useful in determining thatfurther SELEX rounds beyond round 11 did not improve binding.

[0118] Typical data from a similar set of binding competitionexperiments done for latter rounds of HGF SELEX 1 are summarized inTable 1.

[0119] Cloning and sequence analysis of HGF SELEXes 1, 2 and 3.Following determination of pool binding affinities for HGF, we subjectedthe optimal SELEX pools to cloning and sequencing in order to isolateand characterize individual aptamers. Data from 30N7 HGF SELEXes 1 and 3are summarized in Table 2, including binding affinities for many of theaptamers. A similar data set was generated for 30N8 HGF SELEX 2 (Table3). Sequences from HGF SELEX 1, 2 and 3 are designated 8-seq. number,10-seq. number, and 11-seq. number, respectively, referring to the totalnumber of SELEX rounds each cloned pool was subjected to. Sequences wereanalyzed and organized into groups with significant homology. Motifswere analyzed and predicted structures were drawn in order to analyzekey features responsible for binding to HGF.

[0120] Inhibition of HGF-mediated stimulation of cell proliferation.HGF, while not a potent mitogen, does stimulate moderate proliferationof many cell lines, which can be measured by incorporation of³H-thymidine. We assayed the inhibitory activity of HGF aptamers bymeasuring their effect on proliferation of human umbilical veinendothelial cells (HUVECs), or monkey bronchial epithelial (4MBr-5)cells. Based on the binding data and sequence family analysis, fourteenaptamers were chosen for analysis in vitro because they bind to HGF withhigh affinity and are representative of different sequence families. Thesequences are shown in Table 4 aligned by a rough consensus whichcontains bases in common to several families. All sequences are 30N7except 10-2 which is 30N8.

[0121] HGF stimulates proliferation of HUVECs by about two-to-three-fold(data not shown). The initial experiment indicated that aptamers 8-17,8-102, 8-104, 8-122, 8-126, 10-2 and 11-208 were effective inhibitors ofHGF-induced HUVEC proliferation with K_(l) values in the low nanomolarrange (FIG. 6). Aptamers 8-113 and 11-222 were less effective and 8-151exhibited little or no concentration-dependent inhibition. The latterobservation is consistent with the fact that aptamer 8-151 does not bindHGF with high affinity and actually binds worse than the random pool.

[0122] Several approaches were taken to reduce the length of aptamerswhich retained significant inhibition of HGF: 1) boundary determinationsby biochemical separation of partially hydrolyzed aptamers; 2) sequencemotif analysis and educated guessing; and 3) truncate SELEX.

[0123] Boundaries and truncation. Boundary determinations were done fora subset of aptamers that demonstrated in vitro inhibition of HGFactivity. Using a standard alkaline hydrolysis procedure with5′-end-labeled RNA, we examined the 3′-boundaries of 8-17, 8-102, 8-104,8-126, 10-1, and 10-2. Additionally, 3′-end-labeled RNA was used for5′-boundary experiments with 8-17 and 8-102. These experiments weremostly uninformative, probably because the high degree of non-specificbinding of RNA fragments, regardless of size, obscured the binding oftruncated high-affinity aptamers to HGF. Non-specific binding ofvirtually all fragments gave no boundary information, and reducing theprotein concentration did not help. Instead, we tried to use polyanioniccompetitors tRNA and heparin to eliminate nonspecific binding to revealthe actual boundaries. The competitors reduced non-specific binding, andHGF was predominantly bound only by full-length aptamers, revealing noboundary information beyond the possibility that full-length aptamersare strongly preferred.

[0124] The sole exception was aptamer 8-102 which had a plausible3′-boundary between two possible endpoints which made sense with respectto computer-predicted structures (FIG. 7A). Based on the boundary dataand structural data, two truncates of 8-102 were synthesized andanalyzed for binding to HGF. The sequence of the full-length aptamer andthe two truncates are shown, with fixed regions underlined:

[0125]gggaggacgaugcggcgagugccuguuuaugucaucguccgucgucagacgacucgcccga8-102 SEQ ID NO:12

[0126]ggacgaugcggcgagugccuguuuaugucaucgucc (36 mer) SEQ ID NO:13

[0127]gacgaugcggcgagugccuguuuauguc (28 mer) SEQ ID NO:14

[0128] In binding to HGF, the 36 mer bound almost as well as thefull-length aptamer, while the 28 mer bound no better than random 30N7(FIG. 7B), suggesting that the boundary data were correct.

[0129] Truncation by sequence structure prediction. Several attemptswere made to base truncation on motif analysis and predicted structures,but these did not succeed in producing truncates which retained bindingto HGF. For example, aptamer 8-17 folded into a reasonable predictedstructure which suggested two obvious points of truncation from its3-prime terminus, into a 38 mer or 28 mer (FIG. 8A). However, bindinganalysis revealed that neither of these truncates retained significantbinding to HGF (FIG. 8B). These data suggest either that the predictedstructure is incorrect or that some of the 3-prime region past base 38is critical for high-affinity binding of aptamer 8-17 to HGF. These twohypotheses are not mutually exclusive. Nevertheless, we did not succeedin obtaining a useful truncate of 8-17 by boundary and structuralprediction.

[0130] Truncate SELEX. In order to generate additional short aptamers,we subjected advanced rounds of the earlier SELEXes to additional roundsof truncate SELEX, using the Truncation SELEX method described in U.S.patent application Ser. No. 09/275,850, filed Mar. 24, 1999, entitled“The Truncation SELEX Method”, incorporated herein by reference in itsentirety. Binding of RNaseH cleaved pools was examined to determinewhich were the appropriate rounds to use to initiate truncate SELEX(data not shown). None of the RNaseH-cleaved evolved pools was clearlysuperior to another in binding to HGF, therefore, the pools which hadbeen previously cloned were chosen to use in truncate SELEX. Theencouraging result from this experiment was that after RNaseH treatment,the evolved pools bound better to HGF than did random RNA, suggestingthat even in the absence of the fixed regions, significant bindingaffinity was retained. This observation was sufficient evidence tosuggest that truncate SELEX could enrich for sequences which bound toHGF in the absence of fixed regions.

[0131] Three rounds of hybridization truncate SELEX were done inparallel, using as starting pools HGF SELEX 1 round 8 and HGF SELEX 3round 11. The truncate SELEX rounds were done at equi-molar RNA andprotein, starting at 1 nM and decreasing to 0.5 and 0.1 nM.Signal-to-noise ratios were very high during selection. Subsequentmanipulations were satisfactory even though the amount or recovered RNAwas sub-picomolar.

[0132] To evaluate the progress of the SELEX, binding affinities oftruncate rounds two and three were determined compared to those of theRNaseH-cleaved starting pools (FIG. 9). For both SELEXes, the thirdround pools bound with improved affinity for HGF compared with theearlier rounds. Interestingly, the second rounds did not bind HGF betterthan the staring material. The dissociation constants for the thirdround truncate SELEX pools are 1-2 nM, representing a 2-3 foldimprovement. While the magnitude of this improvement is not large, it isprobably significant since HGF as a target did not easily yield affinityenrichment, probably because of its intrinsically high affinity for RNA.

[0133] The two pools were cloned and sequenced, and binding affinitieswere determined (Table 5). The truncated aptamer with the best bindingaffinity, Tr51, is among several sequences which are novel, that is,they were not found in the clones sequenced from the full-length SELEXpools. The emergence of novel sequences suggests that the truncate SELEXsucceeded in amplifying aptamers which were relatively rare in thefull-length pools. Aptamer Tr51 appeared more frequently than any othersequence, consistent with the observation that it has better bindingaffinity than any other truncate. Other sequences which appearedmultiple times also tend to be those with binding affinities near orbetter than the pool K_(d) of 1-2 nM.

[0134] HGF inhibition by the 36 mer aptamer modified with 40K-PEG. The36 mer derivative of aptamer 8-102 described above was tested forinhibition in vitro in a 4MBr-5 cell proliferation assay (FIG. 10).Although the 36 mer retained high-affinity binding to HGF, it did notretain inhibitory activity in vitro comparable to its parent aptamer8-102 and aptamer 8-17 (FIG. 10).

[0135] In order to improve the activity of the 36 mer, we tested it in aformulation with a 3′-dT cap and 5′-40K PEG. The modified aptamer,designated NX22354, was tested for inhibition of HGF-mediatedproliferation 4MBr-5 cells (FIG. 11A). The data indicate that the 36mer-PEG aptamer inhibits HGF, and that it performs at least as well asthe full-length aptamer 8-17, which had previously exhibited thestrongest inhibition of all aptamers tested. As expected, thenon-PEGylated 36 mer did not inhibit HGF, suggesting that the additionof PEG and/or the 3′-cap contribute to the aptamer's bioactivity. Thisexperiment was also done at lower aptamer concentrations, supporting theprevious result and showing more clearly that 36 mer-PEG aptamer is abetter inhibitor that the 8-17 full-length aptamer (FIG. 11B). Alsotested by this assay was a non-binding aptamer containing a 3′-dT capand 5′-40K PEG, the VEGF aptamer NX1838, which had no effect on HGFstimulation (FIG. 12). In this same experiment, a non-PEGylated versionof NX1838 and the truncate SELEX aptamer Tr51 were shown to have noinhibitory effect on HGF (FIG. 12). This suggests that Tr51, similar tothe 36 mer base aptamer of NX22354, may require 5′-40K-PEG to inhibitHGF function.

[0136] Inhibition of HGF-mediated stimulation of cell migration. HGFreadily stimulates cell movement, hence the name, scatter factor. Weassayed the inhibitory effect of HGF aptamers by measuring their effecton A549 cell migration across a Matrigel coated membrane with 8.0 micronpores as described in Materials and Methods (Table 6). The NX22354aptamer fully inhibited HGF-mediated migration at both 1 and 0.2 μMconcentrations, but at 0.04 μM, the effect was negligible. Themonoclonal antibody control (sample 3) was moderately effective at the 1μg/ml dose, which is above its published EC₅₀ value of 0.1-0.3 μg/ml forinhibition of 4MBr-5 cell proliferation.

[0137] Combined inhibitory effect of HGF and VEGF aptamers onHUVECproliferation. It was reported that VEGF and HGF have an additivestimulatory effect on HLUVEC proliferation (Van Belle 1998, Circulation.97:381-90). We observed this effect when VEGF and HGF were added, singlyand in combination, to HUVECs, and we measured incorporation of³H-thymidine (FIG. 13). As expected, stimulation by HGF was relativelyweak compared with that of VEGF and together, the stimulatory effect wasgreater than that elicited by VEGF alone.

[0138] Based on these curves, we chose to add each cytokine at 10 ng/mlfor optimal stimulation in the aptamer inhibition experiments. We thentested the effect of adding one or both aptamers to thedoubly-stimulated cells in the presence of both growth factors (FIG.14A). We observed that each aptamer partially inhibits the stimulationand that both aptamers result in complete inhibition. Interestingly, themagnitude of the inhibitory effect of each aptamer roughly correspondswith the magnitude of the stimulation conferred by each cytokine. Thisobservation suggests that the stimulatory effect of each cytokine can beinhibited independently, and that the two cytokines stimulate HUVECsindependently.

[0139] The remaining two panels of FIG. 14 (FIG. 14B and FIG. 14C) arecontrols in which each cytokine being administered separately,demonstrating that the HGF and VEGF aptamers do not cross-react, thatis, each aptamer affects only the cytokine against which it wasselected. For the HGF stimulated cells, we observed inhibition by theHGF aptamer NX22354, but not by the VEGF aptamer NX1838 (FIG. 14B).Conversely, stimulation by VEGF was inhibited by the VEGF aptamerNX1838, but was unaffected by the HGF aptamer NX22354 (FIG. 14C).

[0140] These data, along with the fact that HGF, like VEGF, is anangiogenesis factor make it intriguing to consider dual administrationof VEGF and HGF aptamers to treat tumors. Furthermore, the availabilityof aptamers which inhibit other growth factors suggests furthercombinations of the VEGF or the HGF aptamer in combination with otheraptamers, for example, aptamers that inhibit bFGF, platelet-derivedgrowth factor (PDGF), transforming growth factor beta (TGF),keratinocyte growth factor (KGF), and/or their receptors allowing forthe possibility that any combination of these inhibitors may berelevant. The goal is to have an array of aptamer-inhibitors ofcytokines and their receptors and to be able to tailor combinationtreatments for specific disease states.

[0141] 2′-O-methyl-purine substitution of HGF aptamer NX22354. Toimprove the stability and pharmacokinetics of NX22354, we determinedwhich of the 17 2′-OH purines could be replaced. We did this bysynthesizing four partially substituted 2′-O-methyl-purine variants ofthe base sequence of NX22354 followed by analysis as described inMaterials and Methods. The four partially-substituted oligonucleotideswere synthesized with a 1:1 ratio of 2′-O-methyl amidite:2′-OH amidite(Table 7). The data analysis measures the ratios of the selected tounselected RNA at each substituted purine position, based onquantitation of bands from the gel. The data are summarized by position(FIG. 15). At each position, the three unsubstituted aptamers provide animportant comparison, which is expressed as an average of the threeunsubstituted aptamers with standard deviation represented by the errorbars. Points that occur at ratios higher than that of the nearbypositions are likely to require 2′-OH for binding.

[0142] The data strongly indicate that two positions, G5 and A25, do nottolerate 2′-OMe substitution. Two other positions, A3 and G10, show aslight preference above the standard deviation of the unselected RNA.

[0143] The set of OMe aptamers were also examined for binding to HGF(data not shown). The binding data indicate that the OMel and OMe3 bindas well as the parent unsubstituted 36 mer, whereas OMe2 and OMe4 bindless well. This suggests that the substitutions in OMe2 and OMe4 areless well tolerated with respect to HGF binding in solution, consistentwith the fact that OMe2 and OMe4 are substituted at A25 and G5,respectively.

[0144] To confirm these results, two aptamers were synthesized which arefully 2′-O-methyl substituted at the apparently well-toleratedpositions. The sequences are shown below, with the 2′-OH-purines shownunderlined. All other purines have 2′-OMe and the pyrimidines are2′-flouro substituted.

[0145] 4×Sub 2′-OH. GGACGAUGCGGCGAGUGCCUGLTUAUGUCAUCGUCC SEQ ID NO:186

[0146] 2×Sub 2′-OH. GGACGAUGCGGCGAGUGCCUGUWTAUGUCAUCGUCC SEQ ID NO:187

[0147] Sequence 4×Sub 2′-OH contains all four of the 2′-OH-purines inquestion, while 2×Sub 2′-OH has only the two 2′-OH-purines most likelyto be required.

[0148] Binding of these oligomers to HGF was examined compared to theunsubstituted parent and the fully 2′-O-methyl substituted RNA (FIG.16). Based on these binding curves, NX22354 tolerates 2′-OMesubstitution at all purines except G5 and A25 (aptamer 2×Sub 2′-OH)withminimal loss of binding affinity. The other two positions in questionapparently are not required to be 2′-OH since aptamer 4×Sub 2′-OH bindsno better than aptamer 2×Sub 2′-OH.

[0149] Two aptamers have been synthesized with 5′-40K-PEG and a 3′-dTcap: one is fully 2′-O-methyl substituted and the other contains 2′-OHat positions G5 and A25. One of these will presumably supplant NX22354as the lead HGF aptamer for further testing in vitro and in vivo.

[0150] Results—c-met

[0151] c-Met SELEX. In the c-Met plate SELEX experiments, theconcentration of nucleic acids was lowered initially, but then raised inlater rounds so that the ratio of the nucleic acid to protein would bevery high. This was done in order to create conditions of highstringency which may select for higher affinity aptamers. Stringency wasalso applied by increasing the number of washes.

[0152] SELEXpool binding. Binding of SELEX pools to c-met was assessedthrough round 7 (FIG. 17). The binding data indicate that the SELEXresulted in about a 20 fold improvement in K_(d) from 20 nM to 1 nM forboth “a” (40N7) and “b” (30N8) pools.

[0153] Since the c-met protein used in SELEX is an IgG fusion protein,we tested random 40N7 and round 7c RNA pools for binding to human IgG₁and c-met. The binding dissociation constants obtained are as follows:TABLE 8 binding and dissociation constants SELEX round Protein K_(d)random IgG₁ ˜1 μM 7c IgG₁ 23 nM random c-met 100 nM 7c c-met 2 nM

[0154] The affinity of round 7c RNA for both IgG₁ and c-met proteinsimproved about 50-fold. There are several interpretations to thisresult. Aptamers may have been selected which bind with better affinityto both proteins. This assumes that the difference in binding betweenIgG₁ and c-met is due to c-met specific aptamers. However, the twoproteins were made in different cell lines which may have differentglycosylation patterns which could influence binding. Thus, if thedifferences in affinity are due to differences between the free IgG₁protein and the IgG₁ domain in c-met, then there might be few if anyc-met specific aptamers in the round 7 pool.

[0155] In order to address these issues further, random and round 5 RNApools from both libraries were examined for binding to the c-met and KDRproteins (FIG. 18). Both of these proteins were made in the same cellline and contain the same IgG₁-His₆ sequence. Random RNA from bothlibraries binds about the same to each protein (K_(d)=˜50 nM). Round 5from the both libraries of c-met SELEX binds better to c-met than to KDR(˜100-fold better for the 30N8 pool and 3-fold better for the 40N7pool). However, round 5 RNA pools do bind better than random RNA to KDR.These results imply that, while there are probably aptamers which bindto human IgG₁ or (HIS)₆ tag in the round 5 pools, there may also bec-met aptamers.

[0156] Detection of IgG aptamers by PCR. Another approach fordetermining if IgG₁ aptamers are present in the SELEX pools was tosubject them to PCR. Predominant IgG₁ aptamers have been isolated fromN7 type libraries which have a known sequence (Nikos Pagratis and ChinhDang, personal communication). For the PCR, a DNA oligonucleotide:

[0157] ML-124; 5′-ACGAGTTTATCGAAAAAGAACGATGGTTCCAATGGAGCA-3′ SEQ IDNO:188 was used that is complementary to the most prevalent N7-serieshuman IgG₁ aptamer sequence, and differs by only a few bases from mostother IgG₁ aptamers. This PCR primer is the same length as the selectedsequence of the major IgG₁ so that it can tolerate mismatches andhybridize to similar sequences.

[0158] The ML-124 3′-primer:

[0159] ML-34; 5′-CGCAGGATCCTAATACGACTCACTATA-3′ SEQ ID NO:189 was usedwith a 5′-primer containing the T7-promoter sequence present in allcloned aptamers to amplify 40N7 series nucleic acids pools: random, 1a,2a, 3a and 4a (data not shown). Since IgG₁ aptamers have not beenisolated from an N8 type library, this analysis was not done for the30N8 SELEX. PCR of random and c-met SELEX round 1a pools yielded nosignal after 20 cycles. However, rounds 2a, 3a, and 4a had steadilyincreasing signals that were easily detectable after 10 PCR cycles. ThusIgG₁ aptamers appeared relatively early in the 40N7 SELEX experiment.For a negative control, PCR was done with a nucleic acid pool from aSELEX known to lack IgG₁ aptamers. For positive controls, PCR was donewith pools from either an N7-based IgG₁ or CTLA4-IgG₁ SELEX. IgG₁aptamers were first isolated from both of these SELEXes. The negativecontrol had no detectable IgG₁ aptamers after 20 PCR cycles. Thepositive controls had detectable signals after 10 PCR cycles.

[0160] C-met aptamers. The sequences of 19 clones from round 7c-40N7fall into five families with two sequences each, a group with threeunrelated members, and six sequences closely related to known IgG₁aptamer sequences (Table 9). Thus, at least 6 of the 19 clones (32%) arehuman IgG₁ aptamers. This confirms the results of previous analysis thatindicated the presence of IgG₁ aptamers in this SELEX experiment.

[0161] Of the 13 clones sequenced from round 7b-30N8, six are almostidentical, another five are closely related, and two are distinct (Table10).

[0162] Nine clones were tested for binding to c-met or KDR, six from the40N7 series and three from the 30N8 series. These clones were chosen forthe following reasons. Clone 7b-4 is the most frequent clone in family 1and is representative of almost all of the sequences isolated from the7b-30N8 library. Clones 7b-10 and 7b-12 are the two clones from the7b-30N8 library that had different sequences. From the 7c-40N7 pool, thechosen representatives were: family 1 (clone 7c-1), family 2 (clone7c-4), family 3 (clone 7c-23), family 4 (clone 7c-26), family 5 (clone7c-25), and the presumed IgG1 family (clone 7c-3).

[0163] Results are shown for only two clones, including 7c-1 which wasthe only one observed to bind to c-met better than KDR (FIG. 19A). Clone7c-1, which appeared twice in the 40N7 series, may exhibit biphasicbinding behavior with a high affinity binding K_(d) of ˜50 μM and alower affinity binding K_(d) of ˜5 nM. All eight other clones bound toKDR as well as to c-met, including 7c-3, which is shown here asrepresentative example (FIG. 19B). Clone 7c-3 and all others besides7c-1 are presumed to be IgG₁ aptamers.

[0164] In summary, two clones (identical to 7c-1) out of 32 apparentlybind c-met specifically and with high affinity. The remaining clonesappear to be IgG₁ aptamers. TABLE 1 Binding affinities of HGF SELEX 1pools with and without competitor tRNA. RNA pool K_(d) (nM) K_(d) (nM)w/tRNA random 30N7 1.6  550   HGF SELEX 1 Rd.8 0.07 0.35 HGF SELEX 1Rd.9 0.09 0.42

[0165] TABLE 2. HGF 30N7 aptamer sequences and binding affinities. Seq.no.^(a) 30N7 random region^(b) SEQ. ID. No. K_(d) (nM) 8-122 (2,1)CGGUGUGAACCUGUUAUGUCCGCGUACCC 18 0.097 8-108GGGUGUGGACCUGUUUAUGUCCGCGUACCC 19  ND^(c) 8-115AGUGAUCCUAUUUAUGACAUCGCGGGCUGC 20 ND 8-125UGUGAACCUGUUUAUGUCAUCUUUUGUCGU 21 0.075 8-155 (1,1)UGUGAACCUAUUUAUGCCAUCUCGAGUGCC 22 0.093 8-162CGUGAGCCUAUUUAUGUCAUCAUGUCUGUC 23 ND 8-165CGAGAGCCUAUUUAUGUCAUCAUGCCUGUG 24 0.100 8-171CGGGAGCCUUUUUAUGUCAUCAUGUCUGUG 25 0.120 8-114 (4,2)CGUGAGCCUAUUUAUGUCAUCAUGUCUGUG 26 0.071 8-203CGCGAGCCUAUUUAUGUCAUCAUGUCUGUG 27 0.140 8-215CGUGAGCCUAUUUAUGUCAUCAUGUCUGGU 28 0.077 8-217CGUGAGCCUAUUUACGUCAUCAUGUCUGUG 29 ND 8-222UGUGAACCUAUUUAUGCCAUUAUGUCUGUG 30 0.130 8-225CGUGAGCCUAUUUAUGUCAUCAAGUCUGUG 31 ND 8-102CGAGUGCCUGUUUAUGUCAUCGUCCGUCGU 12 0.060 11-9   CGUGAGCCUGUUUAUGACCUCGUCCAUGGC 32 0.074 11-58  CGUGAGCCUAUUUAUGACAUGUCCCUCGAG 33 ND 11-59  CGUGAGCCUGUAUAUGUCAUUGUUCUCCGG 34 0.110 11-57  UGAGUACCUGUUUAUGUCACCACUUUCCCC 35 ND 11-103  UGAUUACCUA UUAUGUCUCGCCCUCUC 36 0.200 11-110  UGAUUACCUAUUUAUGUCAUGCUCCUCCCC 37 0.08611-65   UGAUAACCUGUUUAUGCCAUCGUGCUGGGC 38 0.110 11-167 UGAUAACCUGUUUAUGUCAUCGUGCUGGGC 39 ND 11-201 UGAGAACCUAUUUAUGUCAUCGUGUCUGGC 40 ND 11-162 UGAUAACCUAUUUAUGACGUCGUGGCUCCC 41 ND 11-202  UGGGAACCUAUUUAUGUCAUCUCCGUCCC 42 ND 11-106  CGAUGAUGCCUGUUUAUGUCGAUGUCCCCC 43 0.120 11-158 CGAUAGCCUAUUUAUGACCUCGUCCCCGUG 44 0.170 11-112 CGUGAGCCUAUUUAUGACAUCGUUCUUGGC 45 ND 11-124 CGUGAGCCUAUCUAUGUCAUCGUGUGUGCC 46 ND 11-122 UGAGUACUAUUUAUGUCGUCGUUCGUGCC 47 ND 11-217 CGUGAGCCUUCCAAUGACGUCGUCCUUGGC 48 0.071 8-104GCGACUCAAUCUGAAUCGUCUUGUCCCGUG 49 0.050 11-76  UCAGCGGCGCGAGCCUGUUUAUGUC UGCUG 50 0.076 “consensus”CGUGAGCCUAUUUAUGUCAUCGU-C-UG 51 11-8    UCAGUAUGACU UUUAUAGCA CGUUCGCCC52 0.150 11-153  ACAGGUAGUCU UCUAUAGCA CUUCCUCCCC 53 0.190 11-157 UCAGAAUGACU UUCAUAGCA CGCUUUCCC 54 0.260 11-222  ACAUAAGUCU UCUAUAGCUCGUCCUUUGUG 55 0.077 11-223  UCAGUAUGGCU UCUAUAGC UCGUUCCUCGG 56 0.1208-126 (3,1) GUGACUCAAAAUGGUGAUCCUCG UUUCCGC 57 0.099 8-101GUGACUCAAAAUGGUGAUCCUCGAUUUCCGC 58 0.095 8-105GUGACUCAAAAUGGUGAUCCUCGAUUGCCGC 59 ND 8-103 GCCGAAAAUUCGUCGACAUCUCCCUGUCUG 60 0.120 8-118 GGCGACUUUCCUCCAAUUCUCACCUCUGCA 610.160 8-119 GCCAUUCGAUCGA UUCUCCGCCGGAUCGUG 62 0.110 “consensus”CGUGAGCCUAUUUAUGUCAUCGU-C-UG 51 8-3   (2) AUCCCGCGAC CAGGGCGUUUCUUCCUCGUCC 63 0.130 8-112 (3) UCCCGAAUUUAAGUGCGUU UCCUCCGCGUC 64 0.1308-154 (3) UCCCAAGAUUCAGGGCGUU UCUUCCUCGUC 65 0.120 8-117UCCCAAGUUUCAGGGCGUU UCUUCCUCGUC 15 0.130 8-123 UCCCGAGUUUGAGGGCGUUUCUUCUUCGUC 66 0.210 11-121  UCCCAGUUUCAgGGGCGAU UCCUCUUCGUC 67  ND^(c)8-17  (7,1) GCGGCU CGAUG UCGUCUUAUCCCUUUGCCC 68 0.095 8-16  GCGGGCUCGAUG UCGUCUUAUCCCCUUUGCCCC 69 ND 8-158 CCGGCU CGAUG UCGUCUUACCCCUUUGCCC 70 0.310 11-104  GUUUGAG UGAUG UCGUCUUGUCCCGCCUGC 71 0.09111-111  GUUAGAG UUUUG UCGUCUUGUCCCAUGUG 72 ND 11-163  GCUUGAGUC UUUGAUCGUCUUAUCCCUCGU 73 0.082 11-208  GUUUGAG UGACG AUCGUCUUGUCCCAUGUG 740.060 11-212  GUUUGAG UUAAA CAUCGGUUUUCUCCUG 75 0.075 11-6    GACGCGUUGAUU CAUCGUCUUAUCCUGCUG 76 0.240 11-126  GUUUGGGUCU UGAUCUCGUCUUGUCCCGUG 77 0.170 11-165  gUUGAUAGG AGUCAU CAUCGUCUUGUCCGC 780.073 11-215  GUAGUGAG UUUUCAUU GUCUUGUCCCCGUG 79 0.091 11-151 UGAGUCAUAGUGUUG AUCGUCGUAUCCCGU 80 0.170 11-7    GUGGAGUCAAAUCGUCUUGUCCCUUGUCCU 81 0.110 11-166  GUUUGAG UUCUGACA CGUCUUGUCCCAUGC82 0.079 11-17   GUUAGAGC GUGACAG UCGUCUUAUCCCGGGUCA 83 0.130 8-113 (2)UGAAUUCCUCUGGCUGAAAAU GACUUGUGC 84 0.083 8-60  UGAAUUCCUUUGGCUGAAAAUGACUUGUGC 85 ND 11-221  GCAGAGCGAAAAUCGUCUUGUCCCCGACGC 86 0.062 ORPHANS11-123  GUGACUCAAAAUGGUGAUCCUCGUUUCGC 87 0.090 8-151AGGACUAAUCCCUAAGGAAUAGCUUGCCCG 88 8 8-174 UCGAGCUUCUGAGUUAAACUGGGGCCUCCU 89 0.230 8-160 GUCCCCGAAUUUAAAGUGCGUUUUCCUCCGGG 90 0.15011-203  GGUUUUUCUUUUCUUGUUCUCUUCUUUCCCC 91 0.260 11-224 ACAGCGGCGACUAGCCUGUUCAUGCCUGCC 92 0.110 11-107 GUUCUGUGUGUCCACGUUCUUACCCCUGUG 93 0.140

[0166] TABLE 3 HGF 30N8 aptamer sequences and binding affinities. Seq.no.^(a) 30N8 random region^(b) SEQ. ID. No. K_(d) (nM) 10-28CCUGUUCUGAAC GCAAAAUGGCGUGGUGGC 94 0.860 10-40UGUCGUUAGUUUAUUGACAAGGCCCGAAG 95 0.350 10-52UCUUAUUGUGUCCAGCUUCUCCCUGCAGGC 96 0.160 10-72 UGUGGCACUGUUGUCCACAAGGGCCUCA 97 0.450 10-8  UUGACAAGGUACCUGUUGCCUGGCGUUUCU 980.920 10-76 AGUUAGGCUUUAAAGC ACG AUAAUCAGCA 99 0.170 10-47 GUCAAGAGGAAAUGACACGG CUCCACUUUUA 100 0.390 10-2  (10) GCCUGAGUUAAACAUGACGGUUUGUGACCC 101 0.069 10-3  GCCUGAGUUAAACAUGACGGGUUUUGUGACCCCU 102 0.07210-23 (4) GUCUGAGUUGGACACAACGC AUUGAGACCC 103 0.330 10-24GUCUGAGUUgGUCACAACGC AUUGAGACCC 104  ND^(c) 10-37 GUCUGAGUCCGU AGGGCGAUUUGUGUCCC 105 3.05 10-7 UGCCUUAAGAGCGGAA CUCCCUGACCCACC 106 1.45 10-13GAUCUGUUGGCGU GU CUACCCGACCCUCCU 107 0.720 10-17 AACCCUGUUGGCGU GACGUCCCGACCCUCC 108 0.560 10-36 CGUUAGCAUCUGAACGAUGCCCAGCCUCAA 109 1.9410-62 GUUAGACUCAACAUGAGUCCCAGCCUCAA 110 0.440 10-29 UCUGUUGGCGUCGUUCUCCUGACCCUCCUC 111 1.75 10-48 GAGUUCCCUGUUGAC UCGC UCUCCUGACCC 1120.310 10-16 UACAGCGUGUUGGUCCCGGACGGGGACUUAU 113 0.210 10-11CGCCUGGACCGUUUGUUUAUCCCCGUAGUC 114 0.610 10-18 CGUGAUUCCUACCAUCAGGUACCUAUCUUG 115 0.300 10-1 (2) AGUGAUGUGAGAG CGUGCCUCUAGUCGGUG 1160.094 10-57 CGAGCCUCCUACCGUUU AGGUACC AUCUUG 117 0.140 10-27UUAGCCUCCGACCG UAA GGUCCUUUUCUUG 118 0.830 10-53 GGCCUCCAACCGCUAAAGGUUCCAUUCUUG 119 0.310 10-49 CCCGACCUCCUGUAACUGGUUGA GGCACUA 120 0.24010-31 (2) GGGUUCCUGAUUGACCCUGUCUCUAGACCC 121 1.90 10-58GGGGAGGCCCUUCAGCCGUCUCCUUGACCC 122 0.440 10-63 UGUGAUGUGAGGGCGUGCUUCCUAACGGUG 123 0.190 40N8 “hitchhiker” sequences 10-19UUCAUUAUGCAUCGAACAGUAUACCACAGGUGUUCAUGUG 124 ND 10-35AUCCAAAUUCUGGUCAUGAGGCGCUGCAGAUACUGCUGCG 125 2.33 10-38UCUGCGGACGGUGAGGUUAAGUUGCAACGACUGCUUGGCG 126 7.38 10-42CAGACCGUGCAAACCCCCCUUAGAGGGUUUUGUCAUUUAC 127 ND 10-56CCUUAGGGCUCCCAAAAAUCGGGCC CGUCGGGCCGAUCAC 128 0.280 10-68CGCGGGAUUCUCUGAGGACGAGGCACGUGUGGGUAAUUCG 129 1.00 10-67UCGGGCUUGGAUGUGGACGUGUAUUUCUAGCUGUGUACGC 130 0.640 10-4 UUGGGUCGGGACUCGAAAGGAUUUGAUAGGAUACAUGAAU 131 0.610

[0167] TABLE 4 List of HGF aptamers and their binding affinities whichwere tested in vitro for inhibition of acti- vity. Seq. no. randomregion K_(d) (nM) “con- CGUGAGCCUAUUUAUGUCAUCGU-C-UG sensus” 8-17 GCGGCU CGAUG UCGU CUUAUCCCUUUGCCC 0.095 8-102CGAGUGCCUGUUUAUGUCAUCGUCCGUCGU 0.060 8-104GCGACUCAAUCUGAAUCGUCUUGUCCCGUG 0.050 8-112 UCCCGAAUUUAAGUGCGUUUCCUCCGCGUC 0.130 8-113 UGAAUUCCUCUGGCUGAAAAUGA CUUGUGC 0.083 8-122CGGUGUGAACCUGUUUAUGUCCGCGUACCC 0.097 8-126 GUGACUCAAAAUGGUGAUCCUCGUUUCCGC 0.099 11-8    UCAGUAUGACU UUUAUAGCA CGUUCGCCC 0.150 11-76  UCAGCGGCGCGAGCCUGUUUAUGUC UGCUG 0.076 11-166  GUUUGAG UUCUGACA CGUCUUGUCCCAUGC 0.079 11-208  GUUUGAG UGACG AUCGUCU UGUCCCAUGUG 0.060 11-222 ACAUAAGUCU UCUAUAGC UCGUCCUUUGUG 0.077 10-2*   GCCUGAG UUAAACAUGACGGUUUGUGACCC 0.069 8-151 AGGACUAAUCCCUAAGGAAUAGCUUGCCCG 8

[0168] TABLE 5 HGF truncate SELEX 30N sequences. Trunc Sequence ofrandom region Identity to Seq #^(a) # of hit (G)G-30N-CA full-length^(b)K_(d) (nM SEQ. ID. No. GGACGAUGCGGCGAGUGCCUGUUUAUGUCAUCGUCC NX22354 0.113 Tr7 (5) CGGUGUGAACCUGUUUAUGUCCGCGUACCC 8-122 0.67 132 Tr45 (3)UGGGAACCUAUUUAUGUCAUCUCCGUCCC 11-202  1.7 133 Tr70UGGGAACCUAUUUAUGUCAUCGUCUGUGCC New 2.4 134 Tr6CGUGAGCCUAUUUAUGUCAUCAUGUCUGUG 8-114 9.0 135 Tr20UGUGAACCUGUUUAUGCCAUCUCGAGUCCC New 3.4 136 Tr23UGUGAACCUAUUUAUGCCAUCUCGAGUGCC 8-155  ND^(c) 137 Tr42UGAUAACCUAUUUAUGACGUCGUGGCUCCC 11-162  6.1 138 Tr44AGUGAUCCUAUUUAUGCCGUCGCUUCUCGC New 6.5 139 Tr65AGAGNUCCUAUUUAUGACAUCCCAUGCCCC New 1.4 140 Tr48UGAUCACCUGUUUAUGCCAUCGUUCUGGGC 11-65   1.8 141 Tr28GGUGACCCUUUUUAUGACAUCGCGUCUGGC New 4.0 142 Tr51 (6)AAUCACAGGAAUCAACUUCUAUUCCCGCCC New 0.06 143 Tr67AAUCACAGGAAUCGACUUUUAUUCCUGCCC New ND 144 Tr17 GCGGCUCGAUGUCGUCUUAUCCCUUUGCCC 8-17  3.0 145 Tr27 UCGGCUCGUUGUCGUCUUAUCCCUUUGCCC New ND 146 Tr18GCUGGCUCGAUGUCAGGUUAUCCCUUUGCCC New ND 147 Tr4  (4,2)^(d)GUGACUCAAAAUGGUGAUCCUCGUUUCCGC 8-126 1.4 148 Tr31 (2)UGAAUUCCUCUGGCUGAAAAUGACUUGUGC 8-113 9.2 149 Tr15GUUUGAGUGACGAUCGUCUUGUCCCAUGUG 11-208  8.8 150 Tr1AUUGAUUCACUGCAUCCUUGACUCUUCCCC New 7.3 151 Tr5CAGACGACUCGCCCGAAGGACGAUGCGG New 28 152 Tr14GAGUUAUAUUUCGUCACCCGUUCCUUUGCCC New 2.2 153 Tr59ACAGUUUGUCUUCUAUAGCUCGUCGCCCC New 7.2 154 Tr71UCAGAAUGACUUUCAUAGCUCGCUUUCCCC New 7.7 155

[0169] TABLE 6 Invasion of A549 cells through Matrigel is inhibited byHGF aptamer NX22354. Sample HGF 10 ng/ml Inhibitor Cells migrated 1 − − 40 2 + − 240 3 + mAb^(a), 1 μg/ml 120 4 + NX22354, 1 uM  40 5 +NX22354, 0.2 uM  25 6 + NX22354, 0.04 uM 200

[0170] TABLE 7 Partially 2′-O-methyl substituted variants of NX22354.SEQ. ID. SEQUENCE No. NX22354 GGACGAUGCGGCGAGUGCCUGUUUAUGUCAUCGUCC 13(parent) *** ** * ** *** *   *   * *  *  * HGFOMe1GGACGAUGCGGCGAGUGCCUGUUUAUGUCAUCGUCCg 156 HGFOMe2GGACGAUGCGGCGAGUGCCUGUUUAUGUCAUCGUCCg 157 HGFOMe3GGACGAUGCGGCGAGUGCCUGUUUAUGUCAUCGUCCg 158 HGFOMe4GGACGAUGCGGCGAGUGCCUGUUUAUGUCAUCGUCCg 159

[0171] TABLE 9 40N7 sequences isolated from a plate SELEX on humanc-met. Clone name: (number of isolates). Sequence^(a) SEQ ID NO: FAMILY1: 7C-1: (2) UUUGACUAUGUCUGACGGGUCUGUGGUCAAUUCCGCCCC 160 FAMILY 2 7C-4:(1) AUCCGUGUUGAUGUCCAUAUAACCUUAUCCCGUCGCUCCC 161 7C-5: (1)GUGUUGACUUCUAGCCAGAAUAACAUUUUGUACCCCUCCC 162 FAMILY 3 7C-2: (1)UCGUUGAGCUUUUGAUAGGGCUUGUUCUUCGAGCGUCCC 163 7C-23: (1)UGAUCUUGGGUUUGAUCGUAAUUACUUCACCCUCCGUCCC 164 FAMILY 4 7C-26: (2)CUCCUUUUCCGCUAAACAAGACCACUUUGAGCCCUGCCCC 165 FAMILY 5 7C-25: (1)CCACCUCGUUACGUACUGAUUUUGGCAUCGCAGUUUGCCC 166 7C-27: (1)GGGCACCUCGAUACGUACUGAUUUUGAAUAUCAGUUAGCCCC 167 OTHERS 7C-21: (1)CGAUUCGUCGUAUAGAAAUGAUUUGAAUGCACCUCCUCCC 168 7C-24: (1)UGUGUUUGUGUGUUGUGUUUGUUAUUCCUGUUUGUGUCCU 169 7C-32: (1)UCGGUCGUAAAAAAUCGUUGGUGUCUAUCUAUUGUUCUCCC 170 Presumed IgG₁ apta- mers7C-3: (1) UGCUCCAGAGGAACCAUCGUUUACUUCAUUUAUUCGCCC 171 7C-22: (1)UGCUCCUUAGGAACCAUCGUCUAUAUCCCAUUCUGACUGCC 172 7C-30: (1)UGCUCCUCAGGAACCAUCGUUUUUCCCAUGUCCUUCUGCC 173 7C-29: (3)UGCUCCUUGGAUUACCAAGGAACCAUUUUCCUCUACCCCC 174

[0172] TABLE 10 30N8 sequences isolated from a plate SELEX on hu- manc-met. Clone name: SEQ. (number of ID. isolates). Sequence^(a) NO:FAMILY 1: 7b-1: (4) GUGCUCAUUACGAACUUGACCGAUGCCUA 175 7b-9: (1)GGUGCUCAUUACGAACUUGACCGAAGCCUA 176 7b-18: (1)GGUGCUCAUUACGAACUUGACCGAUGCCUA 177 7b-3: (1) AGUGCUCCAAUGAACUUUGCUCGCUGA178 7b-8: (1) GGUGCUCCGUUUGGAACUUGAUCGGUAGGA 179 7b-7: (1)GUGCUCAUUCAGAACUUGACGUAUAACCA 180 7b-14 (1)GGUGCUCCUUAGGAACUUGACCGUCCGCCA 181 7b-16: (1)GUGGUGCUCCACUAACCAAGUGGAACCUUG 182 consensus: GUGCUC-UU--GAACUUGACCG 183OTHERS: 7b-10: (1) ACGAUAAGUGGGAGUGAGUAAGUUUGAGUA 184 7b-12: (1)CCUAGACCCCCAGGUUCCUCCCCACUAGUC 185

[0173]

1 390 98 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 1 GGGAAAAGCG AAUCAUACAC AAGANNNNNNNNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN NNNNNNNNNN NNNNGCUCCG CCAGAGACCAACCGAGAA 98 41 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 2 UAAUACGACU CACUAUAGGGAAAAGCGAAU CAUACACAAG A 41 24 base pairs nucleic acid single linear RNAAll C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 3 UUCUCGGUUGGUCUCUGGCG GAGC 24 96 base pairs nucleic acid single linear RNA All C′sare 2′-NH2 cytosine All U′s are 2′-NH2 uracil 4 GGGAAAAGCG AAUCAUACACAAGAAUGGUU GGCCUGGGCG CAGGCUUCGA 50 AGACUCGGCG GGAACGGGAA UGGCUCCGCCAGAGACCAAC CGAGAA 96 98 base pairs nucleic acid single linear RNA AllC′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 5 GGGAAAAGCGAAUCAUACAC AAGACAGGCA CUGAAAACUC GGCGGGAACG 50 AAAGUAGUGC CGACUCAGACGCGUGCUCCG CCAGAGACCA ACCGAGAA 98 91 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 6GGGAAAAGCG AAUCAUACAC AAGAAGUCUG GCCAAAGACU CGGCGGGAAC 50 GUAAAACGGCCAGAAUUGCU CCGCCAGAGA CCAACCGAGA A 91 94 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 7GGGAAAAGCG AAUCAUACAC AAGAGUAGGA GGUUCCAUCA CCAGGACUCG 50 GCGGGAACGGAAGGUGAUGS GCUCCGCCAG AGACCAACCG AGAA 94 95 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil8 GGGAAAAGCG AAUCAUACAC AAGAACAAGG AUCGAUGGCG AGCCGGGGAG 50 GGCUCGGCGGGAACGAAAUC UGCUCCGCCA GAGACCAACC GAGAA 95 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil9 GGGAAAAGCG AAUCAUACAC AAGAUUGGGC AGGCAGAGCG AGACCGGGGG 50 CUCGGCGGGAACGGAACAGG AAUGCUCCGC CAGAGACCAA CCGAGAA 97 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil10 GGGAAAAGCG AAUCAUACAC AAGAAAGGGA UGGGAUUGGG ACGAGCGGCC 50 AAGACUCGGCGGGAACGAAG GGUGCUCCGC CAGAGACCAA CCGAGAA 97 96 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil11 GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAACGAAA GUGUCAUGGU 50 AGCAAGUCCAAUGGUGGACU CUGCUCCGCC AGAGACCAAC CGAGAA 96 98 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil12 GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAACGUGA AGUGGGUAGG 50 UAGCUGAAGACGGUCUGGGC GCCAGCUCCG CCAGAGACCA ACCGAGAA 98 99 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil13 GGGAAAAGCG AAUCAUACAC AAGAAAGGGA UGGGAUUGGG ACGAGCGGCC 50 AAGACUCGGCGGGAACGAAG GGUCCGCUCC GCCAGAGACC AACCGAGAA 99 98 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil14 GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAACGAAG UGUGUGAGUA 50 ACGAUCACUUGGUACUAAAA GCCCGCUCCG CCAGAGACCA ACCGAGAA 98 100 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil15 GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAAUCGAA AGUGUACUGA 50 AUUAGAACGGUGGGCCUGCU CAUCGUGCUC CGCCAGAGAC CAACCGAGAA 100 103 base pairs nucleicacid single linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2uracil 16 GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAAUCGUA AUGUGGAUGA 50UAGCACGAUG GCAGYAGUAG UCGGACCGCG CUCCGCCAGA GACCAACCGA 100 GAA 103 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 17 GGGAAAAGCG AAUCAUACAC AAGACAGCGG CGGAGUCAGUGAAAGCGUGG 50 GGGGYGCGGG AGGUCUACCC UGACGCUCCG CCAGAGACCA ACCGAGAA 98 95base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 18 GGGAAAAGCG AAUCAUACAC AAGACGGCUG UGUGUGGUAGCGUCAUAGUA 50 GGAGUCGUCA CGAACCAAGG CGCUCCGCCA GAGACCAACC GAGAA 95 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 19 GGGAAAAGCG AAUCAUACAC AAGACGGCUG UGUGGUGUUGGAGCGUCAUA 50 GUAGGAGUCG UCACGAACCA AGGCGCUCCG CCAGAGACCA ACCGAGAA 98 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 20 GGGAAAAGCG AAUCAUACAC AAGACGAUGC GAGGCAAGAAAUGGAGUCGU 50 UACGAACCCU CUUGCAGUGC GCGGCUCCGC CAGAGACCAA CCGAGAA 97 95base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 21 GGGAAAAGCG AAUCAUACAC AAGACGUGCG GAGCAAAUAGGGGAUCAUGG 50 AGUCGUACGA ACCGUUAUCG CGCUCCGCCA GAGACCAACC GAGAA 95 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 22 GGGAAAAGCG AAUCAUACAC AAGACUGGGG AGCAGGAUAUGAGAUGUGCG 50 GGGCAAUGGA GUCGUGACGA ACCGCUCCGC CAGAGACCAA CCGAGAA 97 95base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 23 GGGAAAAGCG AAUCAUACAC AAGAGUCCGC CCCCAGGGAUGCAACGGGGU 50 GGCUCUAAAA GGCUUGGCUA AGCUCCGCCA GAGACCAACC GAGAA 95 94base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 24 GGGAAAAGCG AAUCAUACAC AAGAGAGAAU GAGCAUGGCCGGGGCAGGAA 50 GUGGGUGGCA ACGGAGGCCA GCUCCGCCAG AGACCAACCG AGAA 94 95base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 25 GGGAAAAGCG AAUCAUACAC AAGAGAUACA GCGCGGGUCUAAAGACCUUG 50 CCCCUAGGAU GCAACGGGGU GGCUCCGCCA GAGACCAACC GAGAA 95 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 26 GGGAAAAGCG AAUCAUACAC AAGAUGAAGG GUGGUAAGAGAGAGUCUGAG 50 CUCGUCCUAG GGAUGCAACG GCAGCUCCGC CAGAGACCAA CCGAGAA 97 99base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 27 GGGAAAAGCG AAUCAUACAC AAGACAAACC UGCAGUCGCGCGGUGAAACC 50 UAGGGUUGCA ACGGUACAUC GCUGUGCUCC GCCAGAGACC AACCGAGAA 9997 base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 28 GGGAAAAGCG AAUCAUACAC AAGAGUGGAC UGGAAUCUUCGAGGACAGGA 50 ACGUUCCUAG GGAUGCAACG GACGCUCCGC CAGAGACCAA CCGAGAA 97 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 29 GGGAAAAGCG AAUCAUACAC AAGAGUGUAC CAAUGGAGGCAAUGCUGCGG 50 GAAUGGAGGC CUAGGGAUGC AACGCUCCGC CAGAGACCAA CCGAGAA 97 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 30 GGGAAAAGCG AAUCAUACAC AAGAGUCCCU AGGGAUGCAACGGGCAGCAU 50 UCGCAUAGGA GUAAUCGGAG GUCGCUCCGC CAGAGACCAA CCGAGAA 97 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 31 GGGAAAAGCG AAUCAUACAC AAGAGCCUAG GGAUGCAACGGCGAAUGGAU 50 AGCGAUGUCG UGGACAGCCA GGUGCUCCGC CAGAGACCAA CCGAGAA 97 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 32 GGGAAAAGCG AAUCAUACAC AAGAAUCGAA CCUAGGGAUGCAACGGUGAA 50 GGUUGUGAGG AUUCGCCAUU AGGCGCUCCG CCAGAGACCA ACCGAGAA 98 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 33 GGGAAAAGCG AAUCAUACAC AAGAGCUAGG GAUGCCGCAGAAUGGUCGCG 50 GAUGUAAUAG GUGAAGAUUG UUGCGCUCCG CCAGAGACCA ACCGAGAA 98 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 34 GGGAAAAGCG AAUCAUACAC AAGAGGACCU AGGGAUGCAACGGUCCGACC 50 UUGAUGCGCG GGUGUCCAAG CUACGCUCCG CCAGAGACCA ACCGAGAA 98 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 35 GGGAAAAGCG AAUCAUACAC AAGAAAGGGA GGAGCUAGAGAGGGAAAGGU 50 UACUACGCGC CAGAAUAGGA UGUGCUCCGC CAGAGACCAA CCGAGAA 97 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 36 GGGAAAAGCG AAUCAUACAC AAGACCAACG UACAUCGCGAGCUGGUGGAG 50 AGUUCAUGAG GGUGUUACGG GGUGCUCCGC CAGAGACCAA CCGAGAA 97 96base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 37 GGGAAAAGCG AAUCAUACAC AAGACCCAAC GUGUCAUCGCGAGCUGGCGG 50 AGAGUUCAUG AGGGUUACGG GUGCUCCGCC AGAGACCAAC CGAGAA 96 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 38 GGGAAAAGCG AAUCAUACAC AAGAGUUGGU GCGAGCUGGGGCGGCGAGAA 50 GGUAGGCGGU CCGAGUGUUC GAAUGCUCCG CCAGAGACCA ACCGAGAA 98 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 39 GGGAAAAGCG AAUCAUACAC AAGACUGGCA AGRAGUGCGUGAGGGUACGU 50 UAGGGGUGUU UGGGCCGAUC GCAUGCUCCG CCAGAGACCA ACCGAGAA 98 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 40 GGGAAAAGCG AAUCAUACAC AAGAUUGGUC GUACUGGACAGAGCCGUGGU 50 AGAGGGAUUG GGACAAAGUG UCAGCUCCGC CAGAGACCAA CCGAGAA 97 99base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 41 GGGAAAAGCG AAUCAUACAC AAGAUGUGAG AAAGUGGCCAACUUUAGGAC 50 GUCGGUGGAC UGYGCGGGUA GGCUCGCUCC GCCAGAGACC AACCGAGAA 9998 base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 42 GGGAAAAGCG AAUCAUACAC AAGACAGGCA GAUGUGUCUGAGUUCGUCGG 50 AGUAGACGUC GGUGGACGCG GAACGCUCCG CCAGAGACCA ACCGAGAA 98 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 43 GGGAAAAGCG AAUCAUACAC AAGAUGUGAU UAGGCAGUUGCAGCCGCCGU 50 GCGGAGACGU GACUCGAGGA UUCGCUCCGC CAGAGACCAA CCGAGAA 97 96base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 44 GGGAAAAGCG AAUCAUACAC AAGAUGCCGG UGGAAAGGCGGGUAGGUGAC 50 CCGAGGAUUC CUACCAAGCC AUGCUCCGCC AGAGACCAAC CGAGAA 96 93base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 45 GGGAAAAGCG AAUCAUACAC AAGAGAGGUG RAUGGGAGAGUGGAGCCCGG 50 GUGACUCGAG GAUUCCCGUG CUCCGCCAGA GACCAACCGA GAA 93 97 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 46 GGGAAAAGCG AAUCAUACAC AAGAGUCAUG CUGUGGCUGAACAUACUGGU 50 GAAAGUUCAG UAGGGUGGAU ACAGCUCCGC CAGAGACCAA CCGAGAA 97 96base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 47 GGGAAAAGCG AAUCAUACAC AAGACCGGGG AUGGUGAGUCGGGCAGUGUG 50 ACCGAACUGG UGCCCGCUGA GAGCUCCGCC AGAGACCAAC CGAGAA 96 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 48 GGGAAAAGCG AAUCAUACAC AAGAACACUA ACCAGGUCUCUGAACGCGGG 50 ACGGAGGUGU GGGCGAGGUG GAAGCUCCGC CAGAGACCAA CCGAGAA 97 99base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 49 GGGAAAAGCG AAUCAUACAC AAGACCGUCU CCCGAGAACCAGGCAGAGGA 50 CGUGCUGAAG GAGCUGCAUC UAGAAGCUCC GCCAGAGACC AACCGAGAA 9999 base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 50 GGGAAAAGCG AAUCAUACAC AAGACCGUCU CCGAGAACCAGGCAGAGGAG 50 GUGCUGAAGG RGCUGGCAUC UACAAGCUCC GCCAGAGACC AACCGAGAA 9996 base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 51 GGGAAAAGCG AAUCAUACAC AAGACCCGCA CAUAAUGUAGGGAACAAUGU 50 UAUGGCGGAA UUGAUAACCG GUGCUCCGCC AGAGACCAAC CGAGAA 96 98base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 52 GGGAAAAGCG AAUCAUACAC AAGACGAUGU UAGCGCCUCCGGGAGAGGUU 50 AGGGUCGUGC GGNAAGAGUG AGGUGCUCCG CCAGAGACCA ACCGAGAA 98 99base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 53 GGGAAAAGCG AAUCAUACAC AAGAGGUACG GGCGAGACGAGAUGGACUUA 50 UAGGUCGAUG AACGGGUAGC AGCUCGCUCC GCCAGAGACC AACCGAGAA 9996 base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 54 GGGAAAAGCG AAUCAUACAC AAGACGGUUG CUGAACAGAACGUGAGUCUU 50 GGUGAGUCGC ACAGAUUGUC CUGCUCCGCC AGAGACCAAC CGAGAA 96 97base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 55 GGGAAAAGCG AAUCAUACAC AAGAACUGAG UAAGGUCUGGCGUGGCAUUA 50 GGUUAGUGGG AGGCUUGGAG UAGGCUCCGC CAGAGACCAA CCGAGAA 97 20base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 56 AAGACUCGGC GGGAACGAAA 20 16 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 57 GGAGUCGUGA CGAACC 16 16 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 58CCUAGGGAUG CAACGG 16 18 base pairs nucleic acid single linear RNA AllC′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 59 RCUGGGAGRG UGGGUGUU18 42 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 60 UGUGNNNNAG UNNNNNNNNN UAGACGUCGGUGGACNNNGC GG 42 21 base pairs nucleic acid single linear RNA All C′sare 2′-NH2 cytosine All U′s are 2′-NH2 uracil 61 GGGNNNGUGA CYCGRGGAYU C21 23 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 62 UGANCNNACU GGUGNNNGNG NAG 23 32base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 63 GUCUCYGAAC NNGGNAGGAN GUGNUGGAGN UG 32 71base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 64 GGGAGGACGA UGCGGNNNNN NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN 50 NNNNNCAGAC GACUCGCCCG A 71 32 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil65 TAATACGACT CACTATAGGG AGGACGATGC GG 32 17 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil66 TCGGGCGAGT CGTCCTG 17 71 base pairs nucleic acid single linear RNAAll C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 67 GGGAGGACGAUGCGGCGCGU AUGUGUGAAA GCGUGUGCAC GGAGGCGUCU 50 ACAAUCAGAC GACUCGCCCG A71 71 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 68 GGGAGGACGA UGCGGGGCAU UGUGUGAAUAGCUGAUCCCA CAGGUAACAA 50 CAGCACAGAC GACUCGCCCG A 71 71 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 69 GGGAGGACGA UGCGGUAAUG UGUGAAUCAA GCAGUCUGAA UAGAUUAGAC50 AAAAUCAGAC GACUCGCCCG A 71 71 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 70 GGGAGGACGAUGCGGAUGUG UGAGUAGCUG AGCGCCCGAG UAUGAWACCU 50 GACUACAGAC GACUCGCCCG A71 71 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 71 GGGAGGACGA UGCGGAAACC UUGAUGUGUGAUAGAGCAUC CCCCAGGCGA 50 CGUACCAGAC GACUCGCCCG A 71 70 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 72 GGGAGGACGA UGCGGUUGAG AUGUGUGAGU ACAAGCUCAA AAUCCCGUUG50 GAGGCAGACG ACUCGCCCGA 70 71 base pairs nucleic acid single linear RNAAll C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 73 GGGAGGACGAUGCGGUAGAG GUAGUAUGUG UGGGAGAUGA AAAUACUGUG 50 GAAAGCAGAC GACUCGCCCG A71 71 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 74 GGGAGGACGA UGCGGAAAGU UAUGAGUCCGUAUAUCAAGG UCGACAUGUG 50 UGAAUCAGAC GACUCGCCCG A 71 71 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 75 GGGAGGACGA UGCGGCACGA AAAACCCGAA UUGGGUCGCC CAUAAGGAUG50 UGUGACAGAC GACUCGCCCG A 71 71 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 76 GGGAGGACGAUGCGGGUAAA GAGAUCCUAA UGGCUCGCUA GAUGUGAUGU 50 GAAACCAGAC GACUCGCCCG A71 71 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 77 GGGAGGACGA UGCGGUAACA ACAAUCAAGGCGGGUUCACC GCCCCAGUAU 50 GAGUGCAGAC GACUCGCCCG A 71 71 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 78 GGGAGGACGA UGCGGUAACA ACAAUCAAGG CGGGUUYACC GCCCCAGUAU50 GAGUACAGAC GACUCGCCCG A 71 71 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 79 GGGAGGACGAUGCGGUAACA ACAAUCAAGG CGGGUUYACC GCUCCAGUAU 50 GAGUACAGAC GACUCGCCCG A71 71 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 80 GGGAGGACGA UGCGGUAACA ACAAUCAAGGCGGGUUCACC GCCCCAGUAU 50 GAGUGCAGAC GACUCGCCCG A 71 71 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 81 GGGAGGACGA UGCGGACCAA GCAAUCUAUG GUCGAACGCU ACACAUGAAU50 GACGUCAGAC GACUCGCCCG A 71 71 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 82 GGGAGGACGAUGCGGGAACA UGAAGUAAUC AAAGUCGUAC CAAUAUACAG 50 GAAGCCAGAC GACUCGCCCG A71 70 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 83 GGGAGGACGA UGCGGGACAU GAAGUAAGACCGUCACAAUU CGAAUGAUUG 50 AAUACAGACG ACUCGCCCGA 70 72 base pairs nucleicacid single linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2uracil 84 GGGAGGACGA UGCGGGAACA UGAAGUAAAA AGUCGACGAA UUAGCUGUAA 50CCAAAACAGA CGACUCGCCC GA 72 71 base pairs nucleic acid single linear RNAAll C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 85 GGGAGGACGAUGCGGGAACA UGAAGUAAAA GUCUGAGUUA GUAAAUUACA 50 GUGAUCAGAC GACUCGCCCG A71 72 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 86 GGGAGGACGA UGCGGGAACU UGAAGUUGAANUCGCUAAGG UUAUGGAUUC 50 AAGAUUCAGA CGACUCGCCC GA 72 71 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 87 GGGAGGACGA UGCGGAACAU GAAGUAAUAA GUCGACGUAA UUAGCUGUAA50 CUAAACAGAC GACUCGCCCG A 71 70 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 88 GGGAGGACGAUGCGGAACAU GAAGUAAAAG UCUGAGUUAG AAAUUACAAG 50 UGAUCAGACG ACUCGCCCGA 7071 base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 89 GGGAGGACGA UGCGGUAACA UAAAGUAGCG CGUCUGUGAGAGGAAGUGCC 50 UGGAUCAGAC GACUCGCCCG A 71 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil90 GGGAGGACGA UGCGGAUAGA ACCGCAAGGA UAACCUCGAC CGUGGUCAAC 50 UGAGACAGACGACUCGCCCG A 71 70 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 91 GGGAGGACGA UGCGGUAAGAACCGCUAGCG CACGAUCAAA CAAAGAGAAA 50 CAAACAGACG ACUCGCCCGA 70 71 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 92 GGGAGGACGA UGCGGUUCUC UCCAAGAACY GAGCGAAUAAACSACCGGAS 50 UCACACAGAC GACUCGCCCG A 71 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil93 GGGAGGACGA UGCGGUGUCU CUCCUGACUU UUAUUCUUAG UUCGAGCUGU 50 CCUGGCAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 94 GGGAGGACGA UGCGGCCGUACAUGGUAARC CUCGAAGGAU UCCCGGGAUG 50 AUCCCCAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 95 GGGAGGACGA UGCGGUCCCA GAGUCCCGUG AUGCGAAGAAUCCAUUAGUA 50 CCAGACAGAC GACUCGCCCG A 71 70 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil96 GGGAGGACGA UGCGGGAUGU AAAUGACAAA UGAACCUCGA AAGAUUGCAC 50 ACUCCAGACGACUCGCCCGA 70 72 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 97 GGGAGGACGA UGCGGAUGUAAAUCUAGGCA GAAACGUAGG GCAUCCACCG 50 CAACGACAGA CGACUCGCCC GA 72 70 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 98 GGGAGGACGA UGCGGAUAAC CCAAGCAGCN UCGAGAAAGAGCUCCAUAGA 50 UGAUCAGACG ACUCGCCCGA 70 71 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 99GGGAGGACGA UGCGGCAAAG CACGCGUAUG GCAUGAAACU GGCANCCCAA 50 GUAAGCAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 100 GGGAGGACGA UGCGGCAAAAGGUUGACGUA GCGAAGCUCU CAAAAUGGUC 50 AUGACCAGAC GACUCGCCCG A 71 70 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 101 GGGAGGACGA UGCGGAAGUG AAGCUAAAGC GGAGGGCCAUUCAGUUUCNC 50 ACCACAGACG ACUCGCCCGA 70 70 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 102GGGAGGACGA UGCGGAAGUG AAGCUAAAGS GGAGGGCCAC UCAGAAACGC 50 ACCACAGACGACUCGCCCGA 70 71 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 103 GGGAGGACGA UGCGGCACCGCUAAGCAGUG GCAUAGCCCA GUAACCUGUA 50 AGAGACAGAC GACUCGCCCG A 71 67 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 104 GGGAGGACGA UGCGGCACGC UAAGCAGUGG CAUAGCGWAACCUGUAAGAG 50 ACAGACGACU CGCCCGA 67 71 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 105GGGAGGACGA UGCGGAGAUU ACCAUAACCG CGUAGUCGAA GACAUAUAGU 50 AGCGACAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 106 GGGAGGACGA UGCGGACUCGGGUAGAACGC GACUUGCCAC CACUCCCAUA 50 AAGACCAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 107 GGGAGGACGA UGCGGUCAGA ACUCUGCCGC UGUAGACAAAGAGGAGCUUA 50 GCGAACAGAC GACUCGCCCG A 71 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil108 GGGAGGACGA UGCGGAAUGA GCAUCGAGAG AGCGCGAACU CAUCGAGCGU 50 ACUAACAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 109 GGGAGGACGA UGCGGCAAAGCACGCGUAUG GCAUGAAACU GGCANCCCAA 50 GUAAGCAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 110 GGGAGGACGA UGCGGGAUGC AGCAACCUGA AAACGGCGUCCACAGGUAAU 50 AACAGCAGAC GACUCGCCCG A 71 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil111 GGGAGGACGA UGCGGAAACU CGCUACAAAC ACCCAAUCCU AGAACGUUAU 50 GGAGACAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 112 GGGAGGACGA UGCGGCUAGCAUAGCCACCG GAACAGACAG AUACGAGCAC 50 GAUCACAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 113 GGGAGGACGA UGCGGGAUUC GGAGUACUGA AAAACAACCCUCAAAAGUGC 50 AUAGGCAGAC GACUCGCCCG A 71 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil114 GGGAGGACGA UGCGGGUCCA GGACGGACCG CAGCUGUGAU ACAAUCGACU 50 UACACCAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 115 GGGAGGACGA UGCGGAAACUCGCUACAAAC ACCCAAUCCU AGAACGUUAU 50 GGAGACAGAC GACUCGCCCG A 71 70 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 116 GGGAGGACGA UGCGGCGGCC CUUAUCGGAG GUCUGCGCCACUAAUUACAU 50 CCACCAGACG ACUCGCCCGA 70 67 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 117GGGAGGACGA UGCGGUCCAG AGCGUGAAGA UCAACGUCCC GGNGUCGAAG 50 ACAGACGACUCGCCCGA 67 8 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 118 AUGUGUGA 8 15 base pairsnucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′s are2′-NH2 uracil 119 CAACAAUCAU GAGUR 15 21 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 120AACAUGAAGU AAGUCARUUA G 21 11 base pairs nucleic acid single linear RNAAll C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 121 AGAACCGCWA G11 7 base pairs nucleic acid single linear RNA All C′s are 2′-NH2cytosine All U′s are 2′-NH2 uracil 122 UCUCUCC 7 10 base pairs nucleicacid single linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2uracil 123 CGAAGAAUYC 10 8 base pairs nucleic acid single linear RNA AllC′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 124 AUGUAAAU 8 8 basepairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosine All U′sare 2′-NH2 uracil 125 AACCCAAG 8 80 base pairs nucleic acid singlelinear DNA 126 CTACCTACGA TCTGACTAGC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50NNNNNNNNNN GCTTACTCTC ATGTAGTTCC 80 20 base pairs nucleic acid singlelinear DNA 127 CTACCTACGA TCTGACTAGC 20 25 base pairs nucleic acidsingle linear DNA N AT POSITION 2 AND 4 IS BIOTIN 128 ANANAGGAACTACATGAGAG TAAGC 25 80 base pairs nucleic acid single linear DNA 129CTACCTACGA TCTGACTAGC GGAACACGTG AGGTTTACAA GGCACTCGAC 50 GTAAACACTTGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA130 CTACCTACGA TCTGACTAGC CCCCGAAGAA CATTTTACAA GGTGCTAAAC 50 GTAAAATCAGGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA131 CTACCTACGA TCTGACTAGC GGCATCCCTG AGTCATTACA AGGTTCTTAA 50 CGTAATGTACGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA132 CTACCTACGA TCTGACTAGC TGCACACCTG AGGGTTACAA GGCGCTAGAC 50 GTAACCTCTCGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA133 CTACCTACGA TCTGACTAGC CACGTTTCAA GGGGTTACAC GAAACGATTC 50 ACTCCTTGGCGCTTACTCTC ATGTAGTTCC 80 79 base pairs nucleic acid single linear DNA134 CTACCTACGA TCTGACTAGC CGGACATGAG CGTTACAAGG TGCTAAACGT 50 AACGTACTTGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 135CTACCTACGA TCTGACTAGC CGCATCCACA TAGTTCAAGG GGCTACACGA 50 AATATTGCAGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 136CTACCTACGA TCTGACTAGC TACCCCTTGG GCCTCATAGA CAAGGTCTTA 50 AACGTTAGCGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 137CTACCTACGA TCTGACTAGC CACATGCCTG ACGCGGTACA AGGCCTGGAC 50 GTAACGTTGGCTTACTCTCA TGTAGTTCC 79 80 base pairs nucleic acid single linear DNA 138CTACCTACGA TCTGACTAGC TAGTGCTCCA CGTATTCAAG GTGCTAAACG 50 AAGACGGCCTGCTTACTCTC ATGTAGTTCC 80 79 base pairs nucleic acid single linear DNA139 CTACCTACGA TCTGACTAGC AGCGATGCAA GGGGCTACAC GCAACGATTT 50 AGATGCTCTGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 140CTACCTACGA TCTGACTAGC CCAGGAGCAC AGTACAAGGT GTTAAACGTA 50 ATGTCTGGTGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 141CTACCTACGA TCTGACTAGC ACCACACCTG GGCGGTACAA GGAGTTATCC 50 GTAACGTGTGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 142CTACCTACGA TCTGACTAGC CAAGGTAACC AGTACAAGGT GCTAAACGTA 50 ATGGCTTCGGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 143CTACCTACGA TCTGACTAGC ACCCCCGACC CGAGTACAAG GCATTCGACG 50 TAATCTGGTGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 144CTACCTACGA TCTGACTAGC CAGTACAAGG TGTTAAACGT AATGCCGATC 50 GAGTTGTATGCTTACTCTCA TGTAGTTCC 79 81 base pairs nucleic acid single linear DNA 145CTACCTACGA TCTGACTAGC ACAACGAGTA CAAGGAGATA GACGTAATCG 50 GCGCAGGTATCGCTTACTCT CATGTAGTTC C 81 79 base pairs nucleic acid single linear DNA146 CTACCTACGA TCTGACTAGC CACGACAGAG AACAAGGCGT TAGACGTTAT 50 CCGACCACGGCTTACTCTCA TGTAGTTCC 79 80 base pairs nucleic acid single linear DNA 147CTACCTACGA TCTGACTAGC AGGGAGAACA AGGTGCTAAA CGTTTATCTA 50 CACTTCACCTGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA148 CTACCTACGA TCTGACTAGC AGGACCAAGG TGTTAAACGG CTCCCCTGGC 50 TATGCCTCTTGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA149 CTACCTACGA TCTGACTAGC TACACAAGGT GCTAAACGTA GAGCCAGATC 50 GGATCTGAGCGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA150 CTACCTACGA TCTGACTAGC GGACAAGGCA CTCGACGTAG TTTATAACTC 50 CCTCCGGGCCGCTTACTCTC ATGTAGTTCC 80 81 base pairs nucleic acid single linear DNA151 CTACCTACGA TCTGACTAGC TACACAAGGG GCCAAACGGA GAGCCAGACG 50 CGGATCTGACAGCTTACTCT CATGTAGTTC C 81 79 base pairs nucleic acid single linear DNA152 CTACCTACGA TCTGACTAGC CGGCTATACN NGGTGCTAAA CGCAGAGACT 50 CGATCAACAGCTTACTCTCA TGTAGTTCC 79 80 base pairs nucleic acid single linear DNA 153CTACCTACGA TCTGACTAGC GAGTAGCCAA GGCGTTAGAC GGAGGGGGAA 50 TGGAAGCTTGGCTTACTCTC ATGTAGTTCC 80 73 base pairs nucleic acid single linear DNA154 CTACCTACGA TCTGACTAGC GAGTAGCCAA GGCGTTAGAC GGAGGGGGAA 50 TGGGCTTACTCTCATGTAGT TCC 73 80 base pairs nucleic acid single linear DNA 155CTACCTACGA TCTGACTAGC GAGTAGCCAA GGCGTTAGAC GGAGGGGGAA 50 TGTGAGCACAGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA156 CTACCTACGA TCTGACTAGC TAGCTCCACA CACAASSCGC RGCACATAGG 50 GGATATCTGGGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA157 CTACCTACGA TCTGACTAGC CATCAAGGAC TTTGCCCGAA ACCCTAGGTT 50 CACGTGTGGGGCTTACTCTC ATGTAGTTCC 80 79 base pairs nucleic acid single linear DNA158 CTACCTACGA TCTGACTAGC CATTCACCAT GGCCCCTTCC TACGTATGTT 50 CTGCGGGTGGCTTACTCTCA TGTAGTTCC 79 80 base pairs nucleic acid single linear DNA 159CTACCTACGA TCTGACTAGC GCAACGTGGC CCCGTTTAGC TCATTTGACC 50 GTTCCATCCGGCTTACTCTC ATGTAGTTCC 80 79 base pairs nucleic acid single linear DNA160 CTACCTACGA TCTGACTAGC CCACAGACAA TCGCAGTCCC CGTGTAGCTC 50 TGGGTGTCTGCTTACTCTCA TGTAGTTCC 79 79 base pairs nucleic acid single linear DNA 161CTACCTACGA TCTGACTAGC CCACCGTGAT GCACGATACA TGAGGGTGTG 50 TCAGCGCATGCTTACTCTCA TGTAGTTCC 79 80 base pairs nucleic acid single linear DNA 162CTACCTACGA TCTGACTAGC CGAGGTAGTC GTTATAGGGT RCRCACGACA 50 CAAARCRGTRGCTTACTCTC ATGTAGTTCC 80 79 base pairs nucleic acid single linear DNA163 CTACCTACGA TCTGACTAGC TGGCGGTACG GGCCGTGCAC CCACTTACCT 50 GGGAAGTGAGCTTACTCTCA TGTAGTTCC 79 81 base pairs nucleic acid single linear DNA 164CTACCTACGA TCTGACTAGC CTCTGCTTAC CTCATGTAGT TCCAAGCTTG 50 GCGTAATCATGGCTTACTCT CATGTAGTTC C 81 79 base pairs nucleic acid single linear DNA165 CTACCTACGA TCTGACTAGC AGCGTTGTAC GGGGTTACAC ACAACGATTT 50 AGATGCTCTGCTTACTCTCA TGTAGTTCC 79 81 base pairs nucleic acid single linear DNA 166CTACCTACGA TCTGACTAGC TGATGCGACT TTAGTCGAAC GTTACTGGGG 50 CTCAGAGGACAGCTTACTCT CATGTAGTTC C 81 81 base pairs nucleic acid single linear DNA167 CTACCTACGA TCTGACTAGC CGAGGATCTG ATACTTATTG AACATAMCCG 50 CACNCAGGCTTGCTTACTCT CATGTAGTTC C 81 73 base pairs nucleic acid single linear DNA168 CTACCTACGA TCTGACTAGC CGATCGTGTG TCATGCTACC TACGATCTGA 50 CTAGCTTACTCTCATGTAGT TCC 73 80 base pairs nucleic acid single linear DNA 169CTACCTACGA TCTGACTAGC GCACACAAGT CAAGCATGCG ACCTTCAACC 50 ATCGACCCGAGCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleic acid single linear DNA170 CTACCTACGA TCTGACTAGC ATGCCAGTGC AGGCTTCCAT CCATCAGTCT 50 GACANNNNNNGCTTACTCT CATGTAGTTCC 80 79 base pairs nucleic acid single linear DNA171 CTACCTACGA TCTGACTAGC CACTTCGGCT CTACTCCACC TCGGTCCTCC 50 ACTCCACAGGCTTACTCTCA TGTAGTTCC 79 80 base pairs nucleic acid single linear DNA172 CTACCTACGA TCTGACTAGC CGCTAACTGA CCCTCGATCC CCCCAAGCCA 50 TCCTCATCGCGCTTACTCTC ATGTAGTTCC 80 90 base pairs nucleic acid single linear DNA173 CTACCTACGA TCTGACTAGC ATCTGACTAG CTCGGCGAGA GTACCCGCTC 50 ATGGCTTCGGCGAATGCCCT GCTTACTCTC ATGTAGTTCC 90 80 base pairs nucleic acid singlelinear DNA 174 CTACCTACGA TCTGACTAGC TCCTGAGACG TTACAATAGG CTGCGGTACT 50GCAACGTGGA GCTTACTCTC ATGTAGTTCC 80 79 base pairs nucleic acid singlelinear DNA 175 CTACCTACGA TCTGACTAGC CGGCAGGGCA CTAACAAGGT GTTAAACGTT 50ACGGATGCCG CTTACTCTCA TGTAGTTCC 79 90 base pairs nucleic acid singlelinear DNA 176 CTACCTACGA TCTGACTAGC TGCACACCGG CCCACCCGGA CAAGGCGCTA 50GACGAAATGA CTCTGTTCTG GCTTACTCTC ATGTAGTTCC 90 79 base pairs nucleicacid single linear DNA 177 CTACCTACGA TCTGACTAGC GACGAAGAGG CCAAGGTGATAACCGGAGTT 50 TCCGTCCGCG CTTACTCTCA TGTAGTTCC 79 79 base pairs nucleicacid single linear DNA 178 CTACCTACGA TCTGACTAGC AAGGACTTAG CTATCCAAGGCACTCGACGA 50 AGAGCCCGAG CTTACTCTCA TGTAGTTCC 79 80 base pairs nucleicacid single linear DNA 179 CTACCTACGA TCTGACTAGC ATGCCCAGTT CAAGGTTCTGACCGAAATGA 50 CTCTGTTCTG GCTTACTCTC ATGTAGTTCC 80 80 base pairs nucleicacid single linear DNA 180 CTACCTACGA TCTGACTAGC GCAGCGTGGC CCTGTTTAGCTCATTTGACC 50 GTTCCATCCG GCTTACTCTC ATGTAGTTCC 80 18 base pairs nucleicacid single linear DNA 181 TACAAGGYGY TAVACGTA 18 8 base pairs nucleicacid single linear DNA 182 GGCCCCGT 8 10 base pairs nucleic acid singlelinear DNA 183 RCACGAYACA 10 7 base pairs nucleic acid single linear DNA184 CTTACCT 7 49 base pairs nucleic acid single linear DNA 185TAGCCAAGGT AACCAGTACA AGGTGCTAAA CGTAATGGCT TCGGCTTAC 49 41 base pairsnucleic acid single linear DNA 186 GTAACCAGTA CAAGGTGCTA AACGTAATGGCTTCGGCTTA C 41 26 base pairs nucleic acid single linear DNA 187CCAGTACAAG GTGCTAAACG TAATGG 26 38 base pairs nucleic acid single linearDNA 188 CGCGGTAACC AGTACAAGGT GCTAAACGTA ATGGCGCG 38 36 base pairsnucleic acid single linear DNA 189 GCGGTAACCA GTACAAGGTG CTAAACGTAATGGCGC 36 50 base pairs nucleic acid single linear DNA 190 ACATGAGCGTTACAAGGTGC TAAACGTAAC GTACTTGCTT ACTCTCATGT 50 44 base pairs nucleicacid single linear DNA 191 CGCGCGTTAC AAGGTGCTAA ACGTAACGTA CTTGCTTACTCGCG 44 26 base pairs nucleic acid single linear DNA 192 GCGTTACAAGGTGCTAAACG TAACGT 26 52 base pairs nucleic acid single linear <Unknown>N at position 1 is an amino modifier C6 dT Nucleotide 51 is an inverted-orientation (3′3′ linkage) phosphoramidite 193 NTAGCCAAGG TAACCAGTACAAGGTGCTAA ACGTAATGGC TTCGGCTTAC 50 TT 52 48 base pairs nucleic acidsingle linear DNA 194 TAGCCATTCA CCATGGCCCC TTCCTACGTA TGTTCTGCGGGTGGCTTA 48 47 base pairs nucleic acid single linear DNA 195 AGCTGGCGGTACGGGCCGTG CACCCACTTA CCTGGGAAGT GAGCTTA 47 29 base pairs nucleic acidsingle linear DNA N at position 1 is an amimo modifier C6 dT Nucleotidenumber 28 is an inverted-orientation (3′3′ linkage) phosphoramidite 196NCCAGTACAA GGTGCTAAAC GTAATGGTT 29 40 base pairs nucleic acid singlelinear DNA 197 TAATACGACT CACTATAGGG AGACAAGAAT AAACGCTCAA 40 24 basepairs nucleic acid single linear DNA 198 GCCTGTTGTG AGCCTCCTGT CGAA 2496 base pairs nucleic acid single linear RNA All C′s are 2′-F cytosineAll U′s are 2′-F uracil 199 GGGAGACAAG AAUAAACGCU CAACGAAUCA GUAAACAUAACACCAUGAAA 50 CAUAAAUAGC ACGCGAGACG UCUUCGACAG GAGGCUCACA ACAGGC 96 95base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 200 GGGAGACAAG AAUAAACGCU CAACGAGUUC ACAUGGGAGCAAUCUCCGAA 50 UAAACAACAC GCKAKCGCAA AUUCGACAGG AGGCUCACAA CAGGC 95 96base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 201 GGGAGACAAG AAUAAACGCU CAACGACCAC AAUACAAACUCGUAUGGAAC 50 ACGCGAGCGA CAGUGACGCA UUUUCGACAG GAGGCUCACA ACAGGC 96 97base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 202 GGGAGACAAG AAUAAACGCU CAACGUCAAG CCAGAAUCCGGAACACGCGA 50 GAAAACAAAU CAACGACCAA UCGAUUCGAC AGGAGGCUCA CAAAGGC 97 97base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 203 GGGAGACAAG AAUAAACNCU CAACGACCAC AAUAACCGGAAAUCCCCGCG 50 GUUACGGAAC ACGCGAACAU GAAUUCGACA GGAGGCUCAC AACAGGC 97 95base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 204 GGGAGACAAG AAUAAACGCU CAACGAACCA CGGGGAAAUCCACCAGUAAC 50 ACGCGAGGCA AACAGACCCU CUUCGACAGG AGGCUCACAA CAGGC 95 97base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 205 GGGAGACAAG AAUAAACGCU CAACGAGCAA AAGUACUCACGGGACCAGGA 50 GAUCAGCAAC ACGCGAGACG AAAUUCGACA GGAGGCUCAC AACAGGC 97 96base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 206 GGGAGACAAG AAUAAACGCU CAACGAGCCA GGAACAUCGACGUCAGCAAA 50 CGCGAGCGCA ACCAGUAACA CCUUCGACAG GAGGCUCACA ACAGGC 96 94base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 207 GGGAGACAAG AAUAAACGCU CAACGCACCA GGAACAACGAGAACCAUCAG 50 UAAACGCGAG CGAUUGCAUG UUCGACAGGA GGCUCACAAC AGGC 94 94base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 208 GGGAGACAAG AAUAAACGCU CAACGCACCA GGAACAACAAGAACCAUCAG 50 UAAGCGCGAG CGAUUGCAUA UUCGACAGGA GGCUCACAAC AGGC 94 101base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 209 GGGAGACAAG AAUAAACGCU CAACGAGCAA GGAACGAAUACAAACCAGGA 50 AACUCAGCAA CACGCGAGCA GUAAGAAUUC GACAGGAGGC UCACAACAGG 100C 101 97 base pairs nucleic acid single linear <Unknown> All C′s are2′-F cytosine All U′s are 2′-F uracil 210 GGGAGACAAG AAUAAACGCUCAACAGUUCA CUCAACCGGC ACCAGACUAC 50 GAUCAGCAUU GGCGAGUGAA CACUUCGACAGGAGGCUCAC AACAGGC 97 96 base pairs nucleic acid single linear RNA AllC′s are 2′-F cytosine All U′s are 2′-F uracil 211 GGGAGACAAG AAUAAACGCUCAACUGGCAA CGGGAUAACA ACAAAUGUCA 50 CCAGCACUAG CGAGACGGAA GGUUCGACAGGAGGCUCACA ACAGGC 96 97 base pairs nucleic acid single linear RNA AllC′s are 2′-F cytosine All U′s are 2′-F uracil 212 GGGAGACAAG AAUAAACGCUCAACGAUGAG CGUGACCGAA GCUAUAAUCA 50 GGUCGAUUCA CCAAGCAAUC UUAUUCGACAGGAGGCUCAC AACAGGC 97 95 base pairs nucleic acid single linear RNA AllC′s are 2′-F cytosine All U′s are 2′-F uracil 213 GGGAGACAAG AAUAAACGCUCAAAGGAUCA CACAAACAUC GGUCAAUAAA 50 UAAGUAUUGA UAGCGGGGAU AUUCGACAGGAGGCUCACAA CAGGC 95 97 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 214 GGGAGACAAG AAUAAACGCUCAACAACCCA ACCAUCUAGA GCUUCGAACC 50 AUGGUAUACA AGGGAACACA AAAUUCGCGGAGGCUCCAAC AGGCGGC 97 96 base pairs nucleic acid single linear RNA AllC′s are 2′-F cytosine All U′s are 2′-F uracil 215 GGGAGACAAG AAUAAACGCUCAAGCGGUCA GAAACAAUAG CUGGAUACAU 50 ACCGCGCAUC CGCUGGGCGA UAUUCGACAGGAGGCUCACA ACAGGC 96 97 base pairs nucleic acid single linear RNA AllC′s are 2′-F cytosine All U′s are 2′-F uracil 216 GGGAGACAAG AAUAAACGCUCAAACAAGAG AGUCAAACCA AGUGAGAUCA 50 GAGCGUUUAG CGCGGAAAGC ACAUUCGACAGGAGGCUCAC AACAGGC 97 96 base pairs nucleic acid single linear RNA AllC′s are 2′-F cytosine All U′s are 2′-F uracil 217 GGGAGACAAG AAUAAACGCUCAAACUCGAC UAGUAAUCAC CCUAGCAUAA 50 AUCUCCUCGA GCACAGACGA UAUUCGACAGGAGGCUCACA ACAGGC 96 94 base pairs nucleic acid single linear RNA AllC′s are 2′-F cytosine All U′s are 2′-F uracil 218 GGGAGACAAG AAUAAACGCUCAAUCAGCAG UAAGCGAUCC UAUAAAGAUC 50 AACUAGCCAA AGAUGACUUA UUCGACAGGAGGCUCACAAC AGGC 94 95 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 219 GGGAGACAAG AAUAAACGCUCAAAAAGACG UAUUCGAUUC GAAACGAGAA 50 AGACUUCAAG UGAGCCCGCA GUUCGACAGGAGGCUCACAA CAGGC 95 49 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 220 CUCAACGAAU CAGUAAACAUAACACCAUGA AACAUAAAUA GCACGCGAG 49 47 base pairs nucleic acid singlelinear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 221CUCAACGAGU UCACAUGGGA GCAAUCUCCG AAUAAACAAC ACGCGAG 47 39 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 222 CUCAACGAAC CACGGGGAAA UCCACCAGUA ACACGCGAG 39 38 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 223 CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 42 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 224 CGCUCAACGA GCCAGGAACA UCGACGUCAG CAAACGCGAG CG 42 35base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 225 CUCAACGAGC CAGGACUACG AUCAGCAAAC GCGAG 35 42base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 226 CUCAACGCAC CAGGAACAAC GAGAACCAUC AGUAAACGCG AG42 42 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 227 CUCAACGCAC CAGGAACAAC AAGAACCAUCAGUAAGCGCG AG 42 40 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 228 CACUCAACCG GCACCAGACUACGAUCAGCA UUGGCGAGUG 40 45 base pairs nucleic acid single linear RNAAll C′s are 2′-F cytosine All U′s are 2′-F uracil 229 GAAUCCGGAACACGCGAGAA AACAAAUCAA CGACCAAUCG AUUCG 45 38 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 7,9, 14, 21 G are 2′-O-methyl guanine 8, 15, 18, 22, 27, 31 A are2′-O-methly adenine 230 CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 38base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 7, 9, 13, 14, 21, 24, 28 G are 2′-O-methyl-guanine8, 15, 18, 22, 27, 30, 31 A are 2′-O-methyl-adenine 231 CUCAACGAGCCAGGAACAUC GACGUCAGCA AACGCGAG 38 38 base pairs nucleic acid singlelinear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 7, 9, 14,21, 36 G are 2′-O-methyl-guanine 8, 15, 18, 22, 27, 31, 37 A are2′-O-methyl-adenine 232 CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 38base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 7, 9, 13, 14, 21, 24, 28, 36 G are2′-O-methyl-guanine 8, 15, 18, 22, 27, 30, 31, 37 A are2′-O-methyl-adenine 233 CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 38base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 7, 9, 14 G are 2′-O-methyl-guanine 8, 15, 18, 27, 31A are 2′-O-methyl-adenine 234 CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG38 38 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 7, 9, 13, 14, 24 G are2′-O-methyl-guanine 8, 15, 18, 22, 27, 31 A are 2′-O-methyl-adenine 235CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 59 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 236CUCAACGAGC AAAAGUACUC ACGGGACCAG GAGAUCAGCA ACACGCGAGA 50 CGAAAUUCG 5943 base pairs nucleic acid single linear RNA All C′s are 2′-F cytosineAll U′s are 2′-F uracil 237 CGCUCAACGA CCACAAUACA AACUCGUAUG GAACACGCGAGCG 43 51 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 238 CGCUCAACUG GCAACGGGAU AACAACAAAUGUCACCAGCA CUAGCGAGAC 50 G 51 41 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 239 UCACUCAACCGGCACCAGAC UACGAUCAGC AUUGGCGAGU G 41 70 base pairs nucleic acid singlelinear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 240GGGAGACAAG AAUAAACGCU CAACGAGCAA GGAACGAAUA CAAACCAGGA 50 AACUCAGCAACACGCGAGCA 70 51 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 241 CUCAACGACC ACAAUAACCGGAAAUCCCCG CGGUUACGGA ACACGCGAAC 50 A 51 69 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 242AGAAUAAACG CUCAACGAUG AGCGUGACCG AAGCUAUAAU CAGGUCGAUU 50 CACCAAGCAAUCUUAUUCG 69 50 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 243 ACGCUCAAAG GAUCACACAAACAUCGGUCA AUAAAUAAGU AUUGAUAGCG 50 52 base pairs nucleic acid singlelinear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 244GCUCAAGCGG UCAGAAACAA UAGCUGGAUA CAUACCGCGC AUCCGCUGGG 50 CG 52 58 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 245 ACCAUCUAGA GCUUCGAACC AUGGUAUACA AGGGAACACAAAAUUCGCGG 50 AGGCUCCA 58 96 base pairs nucleic acid single linear RNAAll C′s are 2′-F cytosine All U′s are 2′-F uracil 246 GGGAGACAAGAUAAACGCUC AAACAAGAGA GUCAAACCAA GUGAGAUCAG 50 AGCGUUUAGC GCGGAAAGCACAUUCGACAG GAGGCUCACA ACAGGC 96 87 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 247 GGGAGACAAGAAUAAACGCU CAAAAAGACG UAUUCGAUUC GAAACGAGAA 50 AGACUUCAAG UGAGCCCGCAGUUCGACAGG AGGCUCA 87 97 base pairs nucleic acid single linear RNA AllC′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 248 GGGAGACAAGAAUAAACGCU CAANNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN NNNNNNNNNNNNNUUCGACA GGAGGCUCAC AACAGGC 97 40 base pairs nucleic acid singlelinear DNA 249 TAATACGACT CACTATAGGG AGACAAGAAT AAACGCTCAA 40 24 basepairs nucleic acid single linear DNA 250 GCCTGTTGTG AGCCTCCTGT CGAA 2497 base pairs nucleic acid single linear RNA All C′s are 2′-NH2 cytosineAll U′s are 2′-NH2 uracil 251 GGGAGACAAG AAUAAACGCU CAAGCCCCAAACGCAAGCGA GCAUCCGCAA 50 CAGGGAAGAA GACAGACGAA UGAUUCGACA GGAGGCUCACAACAGGC 97 97 base pairs nucleic acid single linear RNA All C′s are2′-NH2 cytosine All U′s are 2′-NH2 uracil 252 GGGAGACAAG AAUAAACGCUCAAGCCCCAA ACGCAAGUGA GCAUCCGCAA 50 CAGGGAAGAA GACAGACGAU UGAUUCGACAGGAGGCUCAC AACAGGC 97 98 base pairs nucleic acid single linear RNA AllC′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 253 GGGAGACAAGAAAUAAACNC UCAAGCCCCA AACGCAAGUG AGCAUCCGCA 50 ACAGGGAAGA AGACAGAUGAAUGAUUCGAC AGGAGGCUCA CAACAGGC 98 95 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 254GGGAGACAAG AAUAAACNCU CAAGCCCCAA GCAAGUGAGC AUCCGCAACA 50 GGGAAGAAGACAGACGAGUG AUUCGACAGG AGGCUCACAA CAGGC 95 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil255 GGGAGACAAG AAUAAACNCU CAAGCCCCAA ACGCAAGUGA GCAUCCGCAA 50 CAGGGAAGAAGACAGACGAA UGAUUCGACA GGAGGCUCAC AACAGGC 97 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil256 GGGAGACAAG AAUAAACGCU CAAGCAAAAG GCGUAAAUAC ACCUCCGCAA 50 CUGGGAAGAAGACGCAGGGA CGGUUCGACA GGNGGCUCAC AACAGGC 97 98 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil257 GGGAGACAAG AAUAAACGCU CAAACAGCUA CAAGUGGGAC AACAGGGUAC 50 AGCGGAGAGAAACAUCCAAA CAAGUUCGAC AGGAGGCUCA CAACAGGC 98 95 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil258 GGGAGACAAG AAUAAACGCU CAAAUCAACU AAACAACGCA GUCACGAGAA 50 CGACCGGKCUGACUCCGAAA GUUCGACAGG AGGCUCACAA CAGGC 95 95 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil259 GGGAGACAAG AAUAAACGCU CAAACGAGAG CACCAAGGCA ACAGAUGCAG 50 AAGAAGUGUGCGCGCGCGAA AUUCGACAGG AGGCUCACAA CAGGC 95 98 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil260 GGGAGACAAG AAUAAACGCU CAAUAAGACA ACGAACAGAC AGAAGCGAAA 50 AAGGGGCGCCGCAGCAACAA CAAAUUCGAC AGGAGGCUCA CAACAGGC 98 94 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil261 GGGAGACAAG AAUAAACGCU CAACGUGUAC CACAACAGUU CCACGGAAGC 50 UGGAAUAGGACGCAGAGGAA UUCGACAGGA GGCUCACAAC AGGC 94 94 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil262 GGGAGACAAG AAUAAACGCU CAAACAAAAU UWUGGUGGGC CCCGCAACMG 50 GGRGGRAGRCCGUUGAAGGC UUCGACAGGA GGCUCACAAC AGGC 94 94 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil263 GGGAGACAAG AAUAAACGCU CAAGAUCAUA ACGAGAGGAG AGGGAGAACU 50 ACACGCGCGCGAGGAAAGAG UUCGACAGGA GGCUCACAAC AGGC 94 89 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil264 GGGAGACAAG AAUAAACGCU CAAACACAAA UCGGGCAGGG ACUGGGUUGG 50 GCACGGCAGGGCGCCUUCGA CAGGAGGCUC ACAACAGGC 89 97 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 265GGGAGACAAG AAUAAACGCU CAAGUGGGCU CGGGCCGGAU GUCUACGGGU 50 GUGAAGAAACCCCUAGGGCA GGGUUCGACA GGAGGCUCAC AACAGGC 97 95 base pairs nucleic acidsingle linear <Unknown> All U′s are 2′-NH2 uracil 266 GGGAGACAAGAAUAAACGCU CAAGAUCAGC GGAACUAAGA AAUGGAAGGC 50 UAAGCACCGG GAUCGGGAGAAUUCGACAGG AGGCUCACAA CAGGC 95 95 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 267 GGGAGACAAGAAUAAACGCU CAAUAACAAA GCAGCAAAGU ACCAGAGGAG 50 AGUUGGCAGG GUUUAGGCAGCUUCGACAGG AGGCUCACAA CAGGC 95 95 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 268 GGGAGACAGAAUAAACGCUC AAAGACCAAG GGACAGCAGC GGGGAAAAAC 50 AGAUCACAGC UGUAAGAGGGCUUCGACAGG AGGCUCACAA CAGGC 95 93 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 269 GGGAGACAAGAAUAAACGCU CAAAGUCGGG GAUAGAAACA CACUAAGAAG 50 UGCAUCAGGU AGGAGAUAAUUCGACAGGNG GCUCACAACA GGC 93 95 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 270 GGGAGACAAGAAUAAACGCU CAAGAGUAUC ACACAAACCG GCACGGACUA 50 AGCAGAAGGA GGUACGGAAGAUUCGACAGG AGGCUCACAA CAGGC 95 94 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 271 GGGAGACAAGAAUAAACNCU CAACGAAAUA GAAGGAACAG AAGAAUGGBG 50 AWGNGGGAAA UGGCAACGAAUUCGACAGGN GGCUCACAAC AGGC 94 97 base pairs nucleic acid single linearRNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 272 GGGAGACAAGAAUAAACGCU CAAACGAGAC CCUGGAUACG AGGCUGAGGG 50 AAAGGGAGMM MRRAMCUARRCKCUUCGACA GGAGGCUCAC AACAGGC 97 96 base pairs nucleic acid singlelinear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil 273GGGAGACAAG AAUAAACGCU CAAGAAGGAU ACUUAGGACU ACGUGGGAUG 50 GGAUGAAAUGGGAGAACGGG AGUUCGACAG GAGGCUCACA ACAGGC 96 96 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil274 GGGAGACAAG AAUAAACGCU CAAAACGCAC AAAGUAAGGG ACGGGAUGGA 50 UCGCCCUAGGCUGGAAGGGA ACUUCGACAG GAGGCUCACA ACAGGC 96 96 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil275 GGGAGACAAG AAUAAACGCU CAAGGUGAAC GGCAGCAAGG CCCAAAACGU 50 AAGGCCGGAAACNGGAGAGG GAUUCGACAG GNGGCUCACA ACAGGC 96 96 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil276 GGGAGACAAG AAUAAACGCU CAAUGAUAUA CACGUAAGCA CUGAACCAGG 50 CUGAGAUCCAUCAGUGCCCA GGUUCGACAG GAGGCUCACA ACAGGC 96 94 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil277 GGGAGACAAG AAUAAACGCU CAAGAUCAUA ACGAGAGGAG AGGGAGAACU 50 ACACGCGCGCGAGGAAAGAG UUCGACAGGA GGCUCACAAC AGGC 94 96 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil278 GGGAGACAAG AAUAAACGCU CAAUCAAGUA AGGAGGAAGG GUCGUGACAG 50 AAAAACGAGCAAAAAACGCG AGUUCGACAG GAGGCUCACA ACAGGC 96 93 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil279 GGGAGACAAG AAUAAACGCU CAAAAGGUGC CGGGUUGGAG GGGUAGCAAG 50 AAAUGGCUAGGGCGCASGAU UCGACAGGNG GCUCACAACA GGC 93 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil280 GGGAGACAAG AAUAAACGCU CAACCAACGC GCACCCCGCA GCAAACGAAA 50 UUGGGGAGACAGGUGCAAGA CAGUUCGACA GGAGGCUCAC AACAGGC 97 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil281 GGGAGACAAG AAUAAACKCU CAACAAACAA UAUCGGCGCA GGAAAACGUA 50 GAAACGAAAMGGAGCUGCGY GGAUUCGACA GGAGGCUCAC AACAGGC 97 93 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil282 GGGAGACAAG AAUAAACGCU CAAUGAUAGC ACAGUGUAUA AGAAAACGCA 50 ACACCGCGCGCGGAAAGAGU UCGACAGGAG GCUCACAACA GGC 93 96 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil283 GGGAGACAAG AAUAAACGCU CAAGAUCAUC GCAGUAUCGG AAUCGACCCU 50 CAGUGGGUGACAUGCGGACA AGUUCGACAG GAGGCUCACA ACAGGC 96 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil284 GGGAGACAAG AAUAAACGCU CAAGUACCGG GAAGGGAUGA ACUGGGAUAU 50 GGGAACGGAGGUCAGAGGCA CGAUUCGACA GGAGGCUCAC AACAGGC 97 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil285 GGGAGACAAG AAUAAACGCU CAAGCAAUGG AACGCUAGGA GGGAACAUAA 50 GCAGGGCGAGCGGAGUCGAU AGCUUCGACA GGAGGCUCAC AACAGGC 97 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil286 GGGAGACAAG AAUAAACGCU CAAAACAGAA CUGAUCGGCG CAGGUUGAUA 50 AAGGGGCAGCGCGAAGAUCA CAAUUCGACA GGAGGCUCAC AACAGGC 97 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil287 GGGAGACAAG AAUAAACGCU CAAGGGAAAC GGAAAGGGAC AAGGCGAACA 50 GACGAGAAGUAGACGGAGUA GGAUUCGACA GGAGGCUCAC AACAGGC 97 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil288 GGGAGACAAG AAUAAACGCU CAANNNGAGG AAGGGCACGC AAGGAAACAA 50 AACACAAAGCAGAAGUAGUA AGAUUCGACA GGAGGCUCAC AACAGGC 97 95 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil289 GGGAGACAAG AAUAAACGCU CAAGUACRCA GUGAGCAGAA GCAGAGAGAC 50 UUGGGAUGGGAUGAAAUGGK CUUCGACAGG AGGCUCACAA CAGGC 95 97 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil290 GGGAGACAAG AAUAAACNCU CAACCGACGU GGACDCGCAU CGGCAUCCAG 50 ACCAGGCUGNBCNGCACCAS ACGUUCGACA GGAGGCUCAC AACAGGC 97 11 base pairs nucleic acidsingle linear RNA All C′s are 2′-NH2 cytosine All U′s are 2′-NH2 uracil291 GGGAAGAAGA C 11 66 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 292 GGGAGGACGA UGCGGNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 CAGACGACUC GCCCGA 66 61 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 293 GGGAGGACGA UGCGGGCAAA UUGCAUGCGU UUUCGAGUGC UUGCUCAGAC50 GACUCGCCCG A 61 71 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 294 GGGAGGACGA UGCGGUGCUUAAACAACGCG UGAAUCGAGU UCAUCCACUC 50 CUCCUCAGAC GACUCGCCCG A 71 72 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 295 GGGAGGACGA UGCGGUUAAU UCAGUCUCAA ACGGUGCGUUUAUCGAGCCA 50 CUGAUCWGAC GACUCGCCCG AA 72 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 296GGGAGGACGA UGCGGCUUAG AGCUCAAACG GUGUGACUUU CAAGCCCUCU 50 AUGCCCAGACGACUCGCCCG A 71 61 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 297 GGGAGGACGA UGCGGUACCUCAAAUUGCGU GUUUUCAAGC AGUAUCAGAC 50 GACUCGCCCG A 61 61 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 298 GGGAGGACGA UGCGGACCCU CAAAUAACGU GUCUUUCAAG UUGGUCAGAC50 GACUCGCCCG A 61 61 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 299 GGGAGGACGA UGCGGACCCUCAAAUAGCGU GCAUUUCAAG CUGGUCAGAC 50 GACUCGCCCG A 61 61 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 300 GGGAAGACGA UGCGGCGCUC AAAUAAUGCG UUAAUCGAAU UCGCCCAGAC50 GACUCGCCCG A 61 71 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 301 GGGAGGACGA UGCGGCAAACAAGCUCAAAU GACGUGUUUU UCAAGUCCUU 50 GUUGUCAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 302 GGGAGGACGA UGCGGUAGUA AGUCUCAAAU GUUGCGUUUUUCGAAACACU 50 UACAUCAGAC GACUCGCCCG A 71 62 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 303GGGAGGACGA UGCGGAGACU CAAAUGGUGU GUUUUCAAGC CUCUCCCAGU 50 CGACUCGCCC GA62 63 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 304 GGGAGGACGA UGCGGUGCUC AAAUGAUGCGUUUCUCGAAU CCACCCAGAC 50 GACUCGCCCG AGG 63 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 305GGGAGGACGA UGCGGCCAUC GGUCUUGGGC AACGCGUUUU CGAGUUACCU 50 AUGGUCAGACGACUCGCCCG A 71 70 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 306 GGGAGGACGA UGCGGCCAUCGGUCUUGGGC AACGCGUUUU CGAGUUACCU 50 ACAUCAGACG ACUCGCCCGA 70 61 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 307 GGGAGGACGA UGCGGGACCC UUAGGCAACG UGUUUUCAAGUUGGUCAGAC 50 GACUCGCCCG A 61 71 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 308 GGGAGGACGAUGCGGACGUA GCUCUUAGGC AAUGCGUAUU UCGAAUUAGC 50 UGUGUCAGAC GACUCGCCCG A71 71 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 309 GGGAGGACGA UGCGGAGUCU UAGGCAGCGCGUUUUCGAGC UACUCCAUCG 50 CCAGUCAGAC GACUCGCCCG A 71 71 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 310 GGGAAGACGA UGCGGAAUGC UCUUAGGCAG CGCGUUAAUC GAGCUAGCAC50 AUCCUCAGAC GACUCGCCCG A 71 71 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 311 GGGAGGACGAUGGGGAGUCU UAGGCAGCGC GUUUUCGAGC UACUCCAUCG 50 CCAGUCAGAC GACUCGCCCG A71 71 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 312 GGGAGGACGA UGCGGUAAUC UCUUAGGCAUCGCGUUAAUC GAGAUAGAUC 50 ACCGUCAGAC GACUCGCCCG A 71 71 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 313 GGGAGGACGA UGCGGCAAUG UCHCUUAGGC CACGCGUUAA UCGAGCGUGA50 CUGUCAGACG ACUCGCCCGA G 71 71 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 314 GGGAGGACGAUGCGGCAUGG UCUUAGGCGA CGCGUUUAUA UCGAGUCACC 50 AUGCUCAGAC GACUCGCCCG A71 61 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 315 GGGAGGACGA UGCGGGAUGC UUAGGCGCCGUGUUUUCAAG GCCAUCAGAC 50 GACUCGCCCG A 61 72 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 316GGGAGGACGA UGCGGUAAUU GUCUUAGGCG CCGUGUUAUC AAGGCACAAU 50 UUCCCUCAGACGACUCGCCC GA 72 71 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 317 GGGAAGACGA UGCGGCUACUAGUGUCUUAG GCGGAGUGUU UAUCAAUCCA 50 CACAUCAGAC GACUCGCCCG A 71 61 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 318 GGGAGGACGA UGCGGACUGA CUUAGGCUGC GCGCACUUCGAGCAUCAGAC 50 GACUCGCCCG A 61 70 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 319 GGGAGGACGAUGCGGUGGUG UGUCUUUGGC ACCGCGUAUU UUCGAGGUAC 50 ACAUCAGACG ACUCGCCCGA 7070 base pairs nucleic acid single linear RNA All C′s are 2′-F cytosineAll U′s are 2′-F uracil 320 GGGAGGACGA UGCGGUGGUG UGUCUUUGGC ACCGCGUAUUCUCGAGGUAC 50 ACAUCAGACG ACUCGCCCGA 70 61 base pairs nucleic acid singlelinear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 321GGGAGGACGA UGCGGGCUCU UCAGCAACGU GUUAUCAAGU UAGCCCAGAC 50 GACUCGCCCG A61 71 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 322 GGGAGGACGA UGCGGCGUAA CUUCAGCGGUGUGUUAAUCA AGCCUUACGC 50 CAUCUCAGAC GACUCGCCCG A 71 59 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 323 GAGGACGAUG CGGGCUCUUA AGCAACGUGU UAUCAAGUUA GCCCAGACGA50 CUCGCCCGA 59 71 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 324 GGGAGGACGA UGCGGUCUCAAGCAAUGCGU UUAUCGAAUU ACCGUACGCC 50 UCCGUCAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 325 GGGAGGACGA UGCGGAAAUC UCUUAAGCAG CGUGUAAAUCAAGCUAGAUC 50 UUCGUCAGAC GACUCGCCCG A 71 61 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 326GGGAGGACGA UGCGGUUCUU AAGCAGCGCG UCAAUCGAGC UAACCCAGAC 50 GACUCGCCCG A61 62 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 327 GGGAGGACGA UGCGGAUCUU AAGCAGCGCGUCAAUCGAGC UAACCCAGAC 50 GACUCGCCCG AG 62 75 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 328ACAGCUGAUG ACCAUGAUUA CGCCAAGCUU AAGCAGCGCG UUUUCGAGCU 50 CAUGUUGGUCAGACGACUCG CCCGA 75 71 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 329 GGGAGGACGA UGCGGAGGGUCUUAAGCAGU GUGAUAAUCA AACUACUCUC 50 CGUGUCAGAC GACUCGCCCG A 71 62 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 330 GGGAGGACGA UGCGGGAUCU UAAGCAGUGC GUUAUUCGAACUAUCCCAGA 50 CGACUCGCCC GA 62 70 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 331 GGGAGGACGAUGCGGUGCUA UUCUUAAGCG GCGUGUUUUU CAAGCCAAUA 50 UCAUCAGACG ACUCGCCCGA 7071 base pairs nucleic acid single linear RNA All C′s are 2′-F cytosineAll U′s are 2′-F uracil 332 GGGAGGACGA UGCGGUCUUA AGCGGCGCGA UUUUCGAGCCACCGCAUCCU 50 CCGUGCAGAC GACUCGCCCG A 71 61 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 333GGGAGGACGA UGCGGCCUCU UAAGCGUCGU GUUUUUCAAG CUGGUCAGAC 50 GACUCGCCCG A61 71 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 334 GGGAGGACGA UGCGGAUACC ACCUCUUAAGCGACGUGCAU UUCAAGUCAG 50 AUGGUCAGAC GACUCGCCCG A 71 72 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 335 GGGAGGACGA UGCGGUGCUA UUCUUAAGCG GCGUGUAAAU CAAGCUAGAU50 CAUCGUCAGA CGACUCGCCC GA 72 71 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 336 GGGAGGACGAUGCGGAACGA CUCUUAAGCU GUGCGUUUUC GAACAAGUCG 50 UAACUCAGAC GACUCGCCCG A71 61 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 337 GGGAGGACGA UGCGGCUCUC AUUUWGCGCGUAAAUCGAGC UAGCCCAGAC 50 GACUCGCCCG A 61 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 338GGGAGGACGA UGCGGAGUCW CUCUCCACCA KCGUGUKUUA AUCAAGCUAN 50 UGCCUCAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 339 GGGAGGACGA UGCGGUCUACGGUCUCUCUG GCGGUGCGUA AAUCKAACCA 50 GAUCGCAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 340 GGGAGGACGA UGCGGUDAUU UCYUAAUCHG AGCGUUUAUCUAUCUMAAUK 50 AUCCUCAGAC GACUCGCCCG A 71 62 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 341GGGAGGACGA UGCGGAUCGC AAUMUGUWGC GUUCUCKAAA CAGCCUCAGA 50 CGACUCGCCC GA62 61 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 342 GGGAGGACGA UGCGGUGGUU CUAGGCACGUGUUUUCAAGU GUAAUCAGAC 50 GACUCGCCCG A 61 62 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 343GGGAGGACGA UGCGGAAACA UGUGUUUUCG AAUGUGCUCU CCUCCCCAAA 50 CAACYCCCCC AA62 70 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 344 GGGAGGACGA UGCGGAAGGC CGUGUUAAUCAAGGCUGCAA UAAAUCAUCC 50 UCCCCAGACG ACUCGCCCGA 70 71 base pairs nucleicacid single linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil345 GGGAGGACGA UGCGGAGGAU CGUGUUCAUC AAGAUUGCUC GUUCUUUACU 50 GCGUUCAGACGACUCGCCCG A 71 71 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 346 GGGAGGACGA UGCGGUCAAAGUGAAGAAUG GACAGCGUUU UCGAGUUGCU 50 UCACUCAGAC GACUCGCCCG A 71 71 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 347 GGGAGGACGA UGCGGGGAGA AUGGCCAGCG UUUAUCGAGGUGCUCCGUUA 50 ACCGGCAGAC GACUCGCCCG A 71 61 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 348GGGAGGACGA UGCGGGAGGA AUGGACWGCG UAUAUCGAGU UGCCUCAGAC 50 GACUCGCCCG A61 61 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 349 GGGAGGACGA UGCGGAUCGA UUUCAUGCGUUUUUCGAGUG ACGAUCAGAC 50 GACUCGCCCG A 61 61 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 350GGGAGGACGA UGCGGAGACC CUAAGMGSGU KSUUUUCAAS CUGGUCWGAC 50 GACUCGCCCG A61 71 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 351 GGGAGGACGA UGCGGUUAGC CUACACUCUAGGUUCAGUUU UCGAAUCUUC 50 CACCGCWGAC GACUCGCCCG A 71 61 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 352 GGGAGGACGA UGCGGUUAGG UCAAUGAUCU UAGUUUUCGA UUCGUCAGAC50 GACUCGCCCG A 61 61 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 353 GGGAGGACGA UGCGGACGUGUGUAUCRARU UUUCCGCUGU UUGUGCAGAC 50 GACUCGCCCG A 61 71 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 354 GGGAGGACGA UGCGGACAGG GUUCUUAGGC GGAGUGUUCA UCAAUCCAAC50 CAUGUCAGAC GACUCGCCCG A 71 62 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 355 GGGAGGACGAUGCGGCGAUU UCCACAGUUU GUCUUAUUCC GCAUAUCAGA 50 CGACUCGCCC GA 62 61 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 356 GGGAGGACGA UGCGGAUAYU CAGCUYGUGU KUUUUCDAUCUUCCCCAGAC 50 GACUCGCCCG A 61 61 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 357 GGGAGGACGAUGCGGCACAC GUGUUUUCAA GUGUGCUCCU GGGAUCAGAC 50 GACUCGCCCG A 61 61 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 358 GGGAGGACGA UGCGGCAAUG UGUUUCUCAA AUUGCUUUCUCCCUUCAGAC 50 GACUCGCCCG A 61 71 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 359 GGGAGGACGAUGCGGAUACU ACCGUGCGAA CACUAAGUCC CGUCUGUCCA 50 CUCCUCAGAC GACUCGCCCG A71 66 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 360 GGGAGGACGA UGCGGAUACU AUGUGCGUUCACUAAGUCCC GUCGUCCCCU 50 CAGACGACUC GCCCGA 66 71 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 361GGGAGGACGA UGCGGGUACU AUGUACGAUC ACUAAGCCCC AUCACCCUUC 50 UCACUCAGACNACUCGCCCG A 71 61 base pairs nucleic acid single linear RNA All C′s are2′-F cytosine All U′s are 2′-F uracil 362 GGGAGGACGA UGCGGUUACUAUGUACAUUU ACUAAGACCC AACGUCAGAC 50 GACUCGCCCG A 61 72 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 363 GGGAGGACGA UGCGGUUWCU AUGUWCGCCU UACUAAGUAC CCGUCGACUG50 UCCCAUCAGA CGACUCGCCC GA 72 61 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 364 GGGAAGACGAUGCGGUGUUG AUCAAUGAAU GUCCUCCUCC UACCCCAGAC 50 GACUCGCCCG A 61 61 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 365 GGGAGGACGA UGCGGUGUUU GUCAAUGUCA UGAUUAGUUUUCCCACAGAC 50 GACUCGCCCG A 61 64 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 366 GGGAGGACGAUGCGGCGGUC UUAAGCAGUG UGUCAAUCAA ACUAUCGUCA 50 GACGACUCGC CCGA 64 61base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 367 GGGAGGACGA UGCGGUUCUU AAGCAGCGCG UCAAUCGAGCUAACCCAGAC 50 GACUCGCCCG A 61 66 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 368 GGGAGGACGAUGCGGAAUGR CCCGUUACCA WCAAUGCGCC UCDUUGMCCC 5 0 CAAACAACYC CCCCAA 66 70base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 369 GGGAGGACGA UGCGGAAUYU CGUGYUACGC GUYYYCUAUCCAAUCUACCC 50 CMUCUCCAAU CAGACGACYC 70 61 base pairs nucleic acid singlelinear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 370GGGAGGACGA UGCGGCGCUU ACAAUAAUUC UCCCUGAGUA CAGCUCAGAC 50 GACUCGCCCG A61 71 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 371 GGGAGGACGA UGCGGAACUU CUUAGGCAGCGUGCUAGUCA AGCUAAGUUC 50 CACCUCAGAC GACUCGCCCG A 71 70 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 372 GGGAGGACGA UGCGGCACAA UCUUCGGCAG CGUGCAAGAU CAAGCUAUUG50 UUGUCAGACG ACUCGCCCGA 70 61 base pairs nucleic acid single linear RNAAll C′s are 2′-F cytosine All U′s are 2′-F uracil 373 GGGAGGACGAUGCGGUCAUU AACCAAGAUA UGCGAAUCAC CUCCUCAGAC 50 GACUCGCCCG A 61 62 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 374 GGGAGGACGA UGCGGUCAUU CUCUAAAAAA GUAUUCCGUACCUCCACAGA 50 CGACUCGCCC GA 62 61 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 375 GGGAGGACGAUGCGGGUGAU CUUUUAUGCU CCUCUUGUUU CCUGUCAGAC 50 GACUCGCCCG A 61 71 basepairs nucleic acid single linear RNA All C′s are 2′-F cytosine All U′sare 2′-F uracil 376 GGGAGGACNA UGCGGUCUAG GCAUCGCUAU UCUUUACUGAUAUAAUUACU 50 CCCCUCAGAC GACUCGCCCG A 71 61 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 377GGGAGGACGA UGCGGAGUWW GCNCGGUCCA GUCACAUCCW AUCCCCAGAC 50 GACUCGCCCG A61 62 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 378 GGGAGGACGA UGCGGCUCUC AUAUKGWGURUUYUUCMUUC SRGGCUCAAA 50 CAAYYCCCCC AA 62 61 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 379GGGAGGACGA UGCGGCUUGU UAGUUAAACU CGAGUCUCCA CCCCUCAGAC 50 GACUCGCCCG A61 62 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 380 GGGAGGACGA UGCGGUCUCU WCUVACVUGURUUCACAUUU UCGCYUCAAA 50 CAACYCCCCC AA 62 61 base pairs nucleic acidsingle linear RNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 381GGGAGGACGA UGCGGUURAC AAUGRSSCUC RCCUUCCCWG GUCCUCAGAC 50 GACUCGCCCG A61 71 base pairs nucleic acid single linear RNA All C′s are 2′-Fcytosine All U′s are 2′-F uracil 382 AGGAGGACGA UGCGGUUAUC UGAARCWUGCGUAAMCUARU GUSAAASUGC 50 AACRACRAAC AACYCSCCCA A 71 61 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 383 AGGAAGACGA UGCGGUUCGA UUUAUUUGUG UCAUUGUUCU UCCAUCAGAC50 GACUCGCCCG A 61 35 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 384 GUGAUGACAU GGAUUACGCCAGACGACUCG CCCGA 35 16 base pairs nucleic acid single linear RNA All C′sare 2′-F cytosine All U′s are 2′-F uracil 385 UGCGUGUUUU CAAGCA 16 23base pairs nucleic acid single linear RNA All C′s are 2′-F cytosine AllU′s are 2′-F uracil 386 CUCAAAUUGC GUGUUUUCAA GCA 23 33 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 387 GGUACCUCAA AUUGCGUGUU UUCAAGCAGU AUC 33 33 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 388 GGAGUCUUAG GCAGCGCGUU UUCGAGCUAC UCC 33 71 base pairsnucleic acid single linear RNA All C′s are 2′-F cytosine All U′s are2′-F uracil 389 GGGAGGACGA UGCGGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN50 NNNNNCAGAC GACUCGCCCG A 71 97 base pairs nucleic acid single linearRNA All C′s are 2′-F cytosine All U′s are 2′-F uracil 390 GGGAGACAAGAAUAAACGCU CAANNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN NNNNNNNNNNNNNUUCGACA GGAGGCUCAC AACAGGC 97

What is claimed is:
 1. A nucleic acid ligand to hepatocyte growthfactor/scatter factor (HGF) identified according to the methodcomprising: a) preparing a candidate mixture of nucleic acids; b)contacting the candidate mixture of nucleic acids with HGF, whereinnucleic acids having an increased affinity to HGF relative to thecandidate mixture may be partitioned from the remainder of the candidatemixture; c) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; d) amplifying the increased affinitynucleic acids to yield a mixture of nucleic acids enriched for nucleicacids with relatively higher affinity and specificity for binding toHGF, whereby a nucleic acid ligand of HGF may be identified.
 2. Apurified and isolated non-naturally occurring nucleic acid ligand toHGF.
 3. A purified and non-naturally occurring RNA ligand to HGF whereinsaid ligand is selected from the group consisting of SEQ ID NOS:12-14 inFIG. 7, SEQ ID NOS:15-17 in FIG. 8, SEQ ID NOS:18-93 in Table 2, SEQ IDNOS:94-131 in Table 3, SEQ ID NOS:132-155 in Table 5, and SEQ IDNOS:156-159 in Table
 7. 4. The nucleic acid ligand of claim 1 whereinHGF is associated with a solid support, and wherein steps b)-c) takeplace on the surface of said solid support.
 5. The nucleic acid ligandof claim 4 wherein said solid support is comprised of nitrocellulose. 6.The nucleic acid ligand of claim 1 wherein said candidate mixture ofnucleic acids is comprised of single stranded nucleic acids.
 7. Thenucleic acid ligand of claim 6 wherein said single stranded nucleicacids are ribonucleic acids.
 8. The nucleic acid ligand of claim 6wherein said single stranded nucleic acids are deoxyribonucleic acids.9. The nucleic acid ligand of claim 7 wherein said candidate mixture ofnucleic acids comprises 2′-F (2′-fluoro) modified ribonucleic acids. 10.The purified and isolated non-naturally occurring nucleic acid ligand ofclaim 2 wherein said nucleic acid ligand is single stranded.
 11. Thepurified and isolated non-naturally occurring nucleic acid ligand ofclaim 10 wherein said nucleic acid ligand is RNA.
 12. The purified andisolated non-naturally occurring RNA ligand of claim 11 wherein saidligand is comprised of 2′-fluoro (2′-F) modified nucleotides.
 13. Amethod for the treatment of a tumor comprising administering abiologically effective dose of a nucleic acid ligand to HGF.
 14. Amethod for determing the level of HGF in an individual comprising:providing a nucleic acid ligand to HGF; contacting a biological fluidfrom said individual with said nucleic acid ligand; determining theamount of HGF that has bound to said nucleic acid ligand.
 15. A methodfor inhibiting angiogenesis, the method comprising administering abiologically-effective dose of a nucleic acid ligand to HGF.
 16. Apharmaceutical composition for the treatment of a tumor comprising anucleic acid ligand to HGF and a pharmaceutically acceptable excipient.17. A nucleic acid ligand to c-met identified according to the methodcomprising: a) preparing a candidate mixture of nucleic acids; b)contacting the candidate mixture of nucleic acids with c-met, whereinnucleic acids having an increased affinity to c-met relative to thecandidate mixture may be partitioned from the remainder of the candidatemixture; c) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; d) amplifying the increased affinitynucleic acids to yield a mixture of nucleic acids enriched for nucleicacids with relatively higher affinity and specificity for binding toc-met, whereby a nucleic acid ligand of c-met may be identified.
 18. Apurified and isolated non-naturally occurring nucleic acid ligand toc-met.
 19. A purified and non-naturally occurring RNA ligand to HGFwherein said ligand is selected from the group consisting of SEQ IDNOS:160-174 in Table 9 and SEQ ID NOS:175-185 in Table
 10. 20. Thenucleic acid ligand of claim 17 wherein c-met is associated with a solidsupport, and wherein steps b)-c) take place on the surface of said solidsupport.
 21. The nucleic acid ligand of claim 20 wherein said solidsupport is comprised of nitrocellulose.
 22. The nucleic acid ligand ofclaim 17 wherein said candidate mixture of nucleic acids is comprised ofsingle stranded nucleic acids.
 23. The nucleic acid ligand of claim 22wherein said single stranded nucleic acids are ribonucleic acids. 24.The nucleic acid ligand of claim 22 wherein said single stranded nucleicacids are deoxyribonucleic acids.
 25. The nucleic acid ligand of claim23 wherein said candidate mixture of nucleic acids comprises 2′-F(2′-fluoro) modified ribonucleic acids.
 26. The purified and isolatednon-naturally occurring nucleic acid ligand of claim 18 wherein saidnucleic acid ligand is single stranded.
 27. The purified and isolatednon-naturally occurring nucleic acid ligand of claim 26 wherein saidnucleic acid ligand is RNA.
 28. The purified and isolated non-naturallyoccurring RNA ligand of claim 27 wherein said ligand is comprised of2′-fluoro (2′-F) modified nucleotides.
 29. A method for the isolation ofnucleic acid ligands to c-met, comprising: a) preparing a candidatemixture of nucleic acids; b) contacting the candidate mixture of nucleicacids with c-met, wherein nucleic acids having an increased affinity toc-met relative to the candidate mixture may be partitioned from theremainder of the candidate mixture; c) partitioning the increasedaffinity nucleic acids from the remainder of the candidate mixture; d)amplifying the increased affinity nucleic acids to yield a mixture ofnucleic acids enriched for nucleic acids with relatively higher affinityand specificity for binding to c-met, whereby a nucleic acid ligand ofc-met may be identified.
 30. The method of claim 29 wherein saidcandidate mixture comprises single-stranded nucleic acids.
 31. Themethod of claim 30 wherein said single-stranded nucleic acids compriseribonucleic acids.
 32. A method for the treatment of a tumor comprisingadministering a biologically effective dose of a nucleic acid ligand toc-met.
 33. A method for inhibiting angiogenesis, the method comprisingadministering a biologically-effective dose of a nucleic acid ligand toc-met.
 34. A pharmaceutical composition for the treatment of a tumorcomprising a nucleic acid ligand to c-met and a pharmaceuticallyacceptable excipient.
 25. A method for treating a disease in whichelevated HGF is a causative factor, the method comprising administeringa biologically-effective dose of a nucleic acid ligand to HGF.
 36. Amethod for inhibiting tumor development, the method comprisingadministering a biologically effective dose of a nucleic acid ligand toHGF in combination with a biologically effective dose of a nucleic acidligand to vascular endothelial growth factor (VEGF).
 37. A method forinhibiting tumor development, the method comprising administering abiologically effective dose of a nucleic acid ligand to HGF incombination with a biologically effective dose of a nucleic acid ligandto basic fibroblast growth factor (bFGF).
 38. A method for inhibitingtumor development, the method comprising administering a biologicallyeffective dose of a nucleic acid ligand to HGF in combination with abiologically effective dose of a nucleic acid ligand to vascularendothelial growth factor (VEGF) and a biologically effective dose of anucleic acid ligand to basic fibroblast growth factor (bFGF).
 39. Amethod for inhibiting tumor development, the method comprisingadministering biologically effective doses of nucleic acid ligands to atleast two growth factors.
 40. The method of claim 39 wherein said growthfactors are selected from the group consisting of vascular endothelialgrowth factor (VEGF), platelet-derived growth factor (PDGF),transforming growth factor beta (TGFβ), HGF, and keratinocyte growthfactor (KGF).
 41. A method for inhibiting tumor development, the methodcomprising administering biologically effective doses of nucleic acidligands to at least two receptors of growth factors.
 42. The method ofclaim 41 wherein said growth factors are selected from the groupconsisting of vascular endothelial growth factor (VEGF),platelet-derived growth factor (PDGF), transforming growth factor beta(TGFβ), HGF, and keratinocyte growth factor (KGF).
 43. A method ofinhibiting tumor development, the method comprising administeringbiologically-effective doses of nucleic acid ligands to one or morereceptors of growth factors in combination with biologically-effectivedoses of nucleic acid ligands to one or more growth factors.
 44. Themethod of claim 44 wherein said growth factors are selected from thegroup consisting of vascular endothelial growth factor (VEGF),platelet-derived growth factor (PDGF), transforming growth factor beta(TGFβ), HGF, and keratinocyte growth factor (KGF).